AXL expression reflects tumor-immune cell dynamics impacting outcome in non-small cell lung cancer patients treated with immune checkpoint inhibitor monotherapy.

Introduction: AXL receptor expression is proposed to confer immune-checkpoint inhibitor (ICI)-resistance in non-small cell lung cancer (NSCLC) patients. We sought to interrogate AXL expression in conjunction with mutational and tumor-microenvironmental features to uncover predictive mechanisms of resistance in ICI-treated NSCLC patients. Methods: Tumor samples from 111 NSCLC patients treated with ICI-monotherapy were analyzed by immunohistochemistry for tumor- and immune-AXL expression. Subsets of patients were analyzed by whole-exome sequencing (n = 44) and imaging mass cytometry (n = 14). Results were related to ICI-outcome measurements. Results: Tumor-cell AXL expression correlated with aggressive phenotypic features including reduced OS in patients treated with ICIs (P = 0.04) after chemotherapy progression, but conversely associated with improved disease control (P = 0.045) in ICI-treated, PD-L1 high first-line patients. AXL+ immune-cell infiltration correlated with total immune-cell infiltration and improved overall outcomes (PFS: P = 0.044, OS: P = 0.054). Tumor-cell AXL-upregulation showed enrichment in mutations associated with PD-L1-upregulation and ICI-response such as MUC4 and ZNF469, as well as adverse mutations including CSMD1 and LRP1B which associated with an immune-suppressed tumor phenotype and poor ICI prognosis particularly within chemotherapy-treated patients. Tumor mutational burden had no effect on ICI-outcomes and was associated with a lack of tumor-infiltrating immune cells. Spatial-immunophenotyping provided evidence that tumor-cell AXL-upregulation and adverse mutations modulate the tumor microenvironment in favor of infiltrating, activated neutrophils over anti-tumor immune-subsets including CD4 and CD8 T-cells. Conclusion: Tumor-cell AXL-upregulation correlated with distinct oncotypes and microenvironmental immune-profiles that define chemotherapy-induced mechanisms of ICI-resistance, which suggests the combination of AXL inhibitors with current chemoimmunotherapy regimens can benefit NSCLC patients.

A woman in her thirties with confusion. / En kvinne i 30-årene med forvirring.

Background: Coercion is rare in cancer treatment. We present a case where a young woman received gamma knife radiosurgery and immunochemotherapy under compulsory institutional care. Case presentation: A previously healthy patient in her thirties was admitted to hospital due to confusion, apathy, weight loss and sleep disturbance. She had difficulties expressing herself and spoke with considerable latency. A brain MRI revealed a tumour of 23 mm in the left-side centrum semiovale and perifocal oedema, while a CT scan showed a 5.6 cm tumour in the right upper lobe and enlarged mediastinal lymph nodes. She was diagnosed with non-small cell lung cancer, no actionable mutations, PD-L1 <75 %. When she did not wish to return to the hospital to undergo gamma knife radiosurgery, she was readmitted under a formal decision to use coercion, and remained under institutional care for over six months. Today she is fully recovered and has no cancer progression almost five years after diagnosis. Interpretation: This case report illustrates the challenges of brain metastases and use of coercion during cancer treatment, both for the patient and healthcare personnel. There is a need for thorough interdisciplinary discussions and to establish as early as possible a shared understanding of the intention and scope of the forced treatment.

Influence of bariatric surgery on the peripheral blood immune system of female patients with morbid obesity revealed by high-dimensional mass cytometry.

Introduction: Obesity is associated with low-grade chronic inflammation, altered levels of adipocytokines, and impaired regulation of gastrointestinal hormones. Secreted, these factors exert immunostimulatory functions directly influencing peripheral immune cells. Methods: In the realm of this study, we aimed to investigate the composition and activation status of peripheral blood immune cells in female patients with morbid obesity compared to lean controls using high-dimensional mass cytometry. Besides, we also assessed the influence of bariatric surgery with respect to its ability to reverse obesity-associated alterations within the first-year post-surgery. Results: Patients with morbid obesity showed typical signs of chronic inflammation characterized by increased levels of CRP and fibrinogen. Apart from that, metabolic alterations were characterized by increased levels of leptin and resistin as well as decreased levels of adiponectin and ghrelin compared to the healthy control population. All these however, except for ghrelin levels, rapidly normalized after surgery with regard to control levels. Furthermore, we found an increased population of monocytic CD14+, HLA-DR-, CD11b+, CXCR3+ cells in patients with morbid obesity and an overall reduction of the HLA-DR monocytic expression compared to the control population. Although CD14+, HLA-DR-, CD11b+, CXCR3+ decreased after surgery, HLA-DR expression did not recover within 9 - 11 months post-surgery. Moreover, compared to the control population, patients with morbid obesity showed a perturbed CD4+ T cell compartment, characterized by a strongly elevated CD127+ memory T cell subset and decreased naïve T cells, which was not recovered within 9 - 11 months post-surgery. Although NK cells showed an activated phenotype, they were numerically lower in patients with morbid obesity when compared to healthy controls. The NK cell population further decreased after surgery and did not recover quantitatively within the study period. Conclusions: Our results clearly demonstrate that the rapid adaptions in inflammatory parameters and adipocytokine levels that occur within the first year post-surgery do not translate to the peripheral immune cells. Apart from that, we described highly affected, distinct immune cell subsets, defined as CD127+ memory T cells and monocytic CD14+, HLA-DR, CD11b+, CXCR3+ cells, that might play a significant role in understanding and further decoding the etiopathogenesis of morbid obesity.

Comprehensive Characterization of CK1δ-Mediated Tau Phosphorylation in Alzheimer's Disease.

A main pathological event in Alzheimer's disease is the generation of neurofibrillary tangles originating from hyperphosphorylated and subsequently aggregated tau proteins. Previous reports demonstrated the critical involvement of members of the protein kinase family CK1 in the pathogenesis of Alzheimer's disease by hyperphosphorylation of tau. However, precise mechanisms and effects of CK1-mediated tau phosphorylation are still not fully understood. In this study, we analyzed recombinant tau441 phosphorylated by CK1δ in vitro via mass spectrometry and identified ten potential phosphorylation sites, five of them are associated to Alzheimer's disease. To confirm these results, in vitro kinase assays and two-dimensional phosphopeptide analyses were performed with tau441 phosphomutants confirming Alzheimer's disease-associated residues Ser68/Thr71 and Ser289 as CK1δ-specific phosphorylation sites. Treatment of differentiated human neural progenitor cells with PF-670462 and Western blot analysis identified Ser214 as CK1δ-targeted phosphorylation site. The use of an in vitro tau aggregation assay demonstrated a possible role of CK1δ in tau aggregation. Results obtained in this study highlight the potential of CK1δ to be a promising target in the treatment of Alzheimer's disease.

Recent Developments in Mouse Trauma Research Models: A Mini-Review.

The urgency to investigate trauma in a controlled and reproducible environment rises since multiple trauma still account for the most deaths for people under the age of 45. The most common multiple trauma include head as well as blunt thorax trauma along with fractures. However, these trauma remain difficult to treat, partially because the molecular mechanisms that trigger the immediate immune response are not fully elucidated. To illuminate these mechanisms, investigators have used animal models, primarily mice as research subjects. This mini review aims to 1) emphasize the importance of the development of clinically relevant murine trauma research, 2) highlight and discuss the existing conflict between simulating clinically relevant situations and elucidating molecular mechanisms, 3) describe the advantages and disadvantages of established mouse trauma models developed to simulate clinically relevant situations, 4) summarize and list established mouse models in the field of trauma research developed to simulate clinically relevant situations.

Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex.

Thoracic traumas with extra-thoracic injuries result in an immediate, complex host response. The immune response requires tight regulation and can be influenced by additional risk factors such as obesity, which is considered a state of chronic inflammation. Utilizing high-dimensional mass and regular flow cytometry, we define key signatures of obesity-related alterations of the immune system during the response to the trauma. In this context, we report a modification in important components of the splenic response to the inflammatory reflex in obese mice. Furthermore, during the response to trauma, obese mice exhibit a prolonged increase of neutrophils and an early accumulation of inflammation associated CCR2+CD62L+Ly6Chi monocytes in the blood, contributing to a persistent inflammatory phase. Moreover, these mice exhibit differences in migration patterns of monocytes to the traumatized lung, resulting in decreased numbers of regenerative macrophages and an impaired M1/M2 switch in traumatized lungs. The findings presented in this study reveal an attenuation of the inflammatory reflex in obese mice, as well as a disturbance of the monocytic compartment contributing to a prolonged inflammation phase resulting in fewer phenotypically regenerative macrophages in the lung of obese mice.

CK1δ-Derived Peptides as Novel Tools Inhibiting the Interactions between CK1δ and APP695 to Modulate the Pathogenic Metabolism of APP.

Alzheimer's disease (AD) is the major cause of dementia, and affected individuals suffer from severe cognitive, mental, and functional impairment. Histologically, AD brains are basically characterized by the presence of amyloid plaques and neurofibrillary tangles. Previous reports demonstrated that protein kinase CK1δ influences the metabolism of amyloid precursor protein (APP) by inducing the generation of amyloid-ß (Aß), finally contributing to the formation of amyloid plaques and neuronal cell death. We therefore considered CK1δ as a promising therapeutic target and suggested an innovative strategy for the treatment of AD based on peptide therapeutics specifically modulating the interaction between CK1δ and APP. Initially, CK1δ-derived peptides manipulating the interactions between CK1δ and APP695 were identified by interaction and phosphorylation analysis in vitro. Selected peptides subsequently proved their potential to penetrate cells without inducing cytotoxic effects. Finally, for at least two of the tested CK1δ-derived peptides, a reduction in Aß levels and amyloid plaque formation could be successfully demonstrated in a complex cell culture model for AD. Consequently, the presented results provide new insights into the interactions of CK1δ and APP695 while also serving as a promising starting point for further development of novel and highly innovative pharmacological tools for the treatment of AD.

Induction of alveolar and bronchiolar phenotypes in human lung organoids.

Patient-derived organoids have revolutionized biomedical research and therapies by "transferring the patient into the Petri dish". In vitro access to human lung organoids representing distal lung tissue, i.e. alveolar organoids, would facilitate research pertaining to a wide range of medical conditions and might open for a future approach to individualized treatment.We propose a protocol to derive a single human lung biopsy towards both alveolar and bronchiolar organoids. By modulating Wnt pathway, we obtained a differential gene expression of the main markers for both subtypes, such as a higher expression of surfactant protein C in alveolar organoids or a higher expression of mucine 5AC in bronchiolar organoids. Although the specific cell enrichment was not complete, the differentiation was observed as early as passage 1 based on morphology, and confirmed by QPCR and histology at passage 2. These results are consistent with a functional specification of lung epithelium towards both alveoli- and bronchi-enriched organoids from first passages.

Regulation of MHC I Molecules in Glioblastoma Cells and the Sensitizing of NK Cells.

Immunotherapy has been established as an important area in the therapy of malignant diseases. Immunogenicity sufficient for immune recognition and subsequent elimination can be bypassed by tumors through altered and/or reduced expression levels of major histocompatibility complex class I (MHC I) molecules. Natural killer (NK) cells can eliminate tumor cells in a MHC I antigen presentation-independent manner by an array of activating and inhibitory receptors, which are promising candidates for immunotherapy. Here we summarize the latest findings in recognizing and regulating MHC I molecules that affect NK cell surveillance of glioblastoma cells.

Activity-Based Probes to Utilize the Proteolytic Activity of Cathepsin G in Biological Samples.

Neutrophils, migrating to the site of infection, are able to release serine proteases after being activated. These serine proteases comprise cathepsin G (CatG), neutrophil elastase protease 3 (PR3), and neutrophil serine protease 4 (NSP4). A disadvantage of the uncontrolled proteolytic activity of proteases is the outcome of various human diseases, including cardiovascular diseases, thrombosis, and autoimmune diseases. Activity-based probes (ABPs) are used to determine the proteolytic activity of proteases, containing a set of three essential elements: Warhead, recognition sequence, and the reporter tag for detection of the covalent enzyme activity-based probe complex. Here, we summarize the latest findings of ABP-mediated detection of proteases in both locations intracellularly and on the cell surface of cells, thereby focusing on CatG. Particularly, application of ABPs in regular flow cytometry, imaging flow cytometry, and mass cytometry by time-of-flight (CyTOF) approaches is advantageous when distinguishing between immune cell subsets. ABPs can be included in a vast panel of markers to detect proteolytic activity and determine whether proteases are properly regulated during medication. The use of ABPs as a detection tool opens the possibility to interfere with uncontrolled proteolytic activity of proteases by employing protease inhibitors.

Application of an Activity-Based Probe to Determine Proteolytic Activity of Cell Surface Cathepsin G by Mass Cytometry Data Acquisition.

During an immune response, cathepsin G (CatG) takes on the role of adaptive and innate immunity and the outcome depends on the localization of CatG. Soluble, cell surface-bound, or intracellular CatG is also responsible for pathophysiology conditions. We applied the activity-based probe MARS116-Bt to mass cytometry by time-of-flight to analyze CatG activity on the cell surface of immune cells. The phosphonate warhead of MARS116-Bt binds covalently to the serine amino acid residue S195 of the catalytic center and thereby CatG activity can be detected. This method contributes to observing the activation or inhibition status of cells during pathogenesis of diseases and enables accurate data acquisition from complex biological samples with a vast panel of cell subset markers in a single-cell resolution.

Influence of obesity on remodeling of lung tissue and organization of extracellular matrix after blunt thorax trauma.

BACKGROUND: Previously, it has been shown that obesity is a risk factor for recovery, regeneration, and tissue repair after blunt trauma and can affect the rate of muscle recovery and collagen deposition after trauma. To date, lung tissue regeneration and extracellular matrix regulation in obese mice after injury has not been investigated in detail yet. METHODS: This study uses an established blunt thorax trauma model to analyze morphological changes and alterations on gene and protein level in lean or obese (diet-induced obesity for 16 ± 1 week) male C57BL/6 J mice at various time-points after trauma induction (1 h, 6 h, 24 h, 72 h and 192 h). RESULTS: Morphological analysis after injury showed lung parenchyma damage at early time-points in both lean and obese mice. At later time-points a better regenerative capacity of lean mice was observed, since obese animals still exhibited alveoli collapse, wall thickness as well as remaining filled alveoli structures. Although lean mice showed significantly increased collagen and fibronectin gene levels, analysis of collagen deposition showed no difference based on colorimetric quantification of collagen and visual assessment of Sirius red staining. When investigating the organization of the ECM on gene level, a decreased response of obese mice after trauma regarding extracellular matrix composition and organization was detectable. Differences in the lung tissue between the diets regarding early responding MMPs (MMP8/9) and late responding MMPs (MMP2) could be observed on gene and protein level. Obese mice show differences in regulation of extracellular matrix components compared to normal weight mice, which results in a decreased total MMP activity in obese animals during the whole regeneration phase. Starting at 6 h post traumatic injury, lean mice show a 50% increase in total MMP activity compared to control animals, while MMP activity in obese mice drops to 50%. CONCLUSIONS: In conclusion, abnormal regulation of the levels of extracellular matrix genes in the lung may contribute to an aberrant regeneration after trauma induction with a delay of repair and pathological changes of the lung tissue in obese mice.

Downregulation of ORP3 Correlates with Reduced Survival of Colon Cancer Patients with Advanced Nodal Metastasis and of Female Patients with Grade 3 Colon Cancer.

Genome instability is an essential hallmark in tumor development, including colorectal cancer. We have recently identified the oxysterol binding protein-related protein 3 (ORP3), also known as oxysterol binding protein-like 3 (OSBPL3), as a novel ploidy-control gene, whose knock-out leads to aneuploidy induction and promotes tumor formation, indicating that ORP3 is a bona fide tumor suppressor protein. Here we analyzed expression of ORP3 in a cohort (n = 206) of colon cancer patients in relation to patient survival. We show that low ORP3 mRNA levels correlate with reduced survival of patients with advanced nodal metastasis (N2). While patient survival does not associate with grading when the whole cohort is evaluated, importantly, low ORP3 mRNA levels associate with worse survival of female patients with grade 3 colon cancer. Similarly, low ORP3 mRNA levels associate with worse survival of grade 3 colon cancer patients 70 years of age and younger while low ORP3 mRNA levels seem to be beneficial for colon cancer patients with a T2 tumor size. Together, the data show that ORP3 expression is downregulated during colon cancer progression, which correlates with reduced patient survival. Thus, ORP3 mRNA levels may be a prognostic marker for better stratification of colon cancer patients.

Influence of Obesity on the Organization of the Extracellular Matrix and Satellite Cell Functions After Combined Muscle and Thorax Trauma in C57BL/6J Mice.

Obesity has been described as a major factor of health risk in modern society. Next to intricately linked comorbidities like coronary artery disease or diabetes, an influence of obesity on regeneration after muscle injury has been described previously. However, the influence of obesity on tissue regeneration in a combined trauma, merging the more systemic influence of a blunt lung trauma and the local blunt muscle trauma, has not been investigated yet. Therefore, the aim of this study was to investigate the influence of obesity on regeneration in a mouse model that combined both muscle and thorax trauma. Using gene expression analysis, a focus was put on the structure as well as the organization of the extracellular matrix and on functional satellite cell physiology. An increased amount of debris in the lung of obese mice compared to normal weight mice up to 192 h after combined trauma based on visual assessment can be reported which is accompanied by a decreased response of Mmp2 in obese mice. Additionally, a delayed and elongated response of inhibitor genes like Timp1 has been revealed in obese mice. This elongated response to the trauma in obese mice can also be seen in plasma based on increased levels of pro-inflammatory chemo- and cytokines (IL-6, MCP-1, and IL 23) 192 h post trauma. In addition to changes in the lung, morphological analysis of the injured extensor iliotibialis anticus of the left hind leg in lean and diet-induced obese mice revealed deposition of fat in the regenerating muscle in obese animals hindering the structure of a compact muscle. Additionally, decreased activation of satellite cells and changes in organization and build-up of the ECM could be detected, finally leading to a decreased stability of the regenerated muscle in obese mice. Both factors contribute to an attenuated response to the trauma by obese mice which is reflected by a statistically significant decrease in muscle force of obese mice compared to lean mice 192 h post trauma induction.

Structure, regulation, and (patho-)physiological functions of the stress-induced protein kinase CK1 delta (CSNK1D).

Members of the highly conserved pleiotropic CK1 family of serine/threonine-specific kinases are tightly regulated in the cell and play crucial regulatory roles in multiple cellular processes from protozoa to human. Since their dysregulation as well as mutations within their coding regions contribute to the development of various different pathologies, including cancer and neurodegenerative diseases, they have become interesting new drug targets within the last decade. However, to develop optimized CK1 isoform-specific therapeutics in personalized therapy concepts, a detailed knowledge of the regulation and functions of the different CK1 isoforms, their various splice variants and orthologs is mandatory. In this review we will focus on the stress-induced CK1 isoform delta (CK1δ), thereby addressing its regulation, physiological functions, the consequences of its deregulation for the development and progression of diseases, and its potential as therapeutic drug target.

Superbubbles revisited.

BACKGROUND: Superbubbles are distinctive subgraphs in direct graphs that play an important role in assembly algorithms for high-throughput sequencing (HTS) data. Their practical importance derives from the fact they are connected to their host graph by a single entrance and a single exit vertex, thus allowing them to be handled independently. Efficient algorithms for the enumeration of superbubbles are therefore of important for the processing of HTS data. Superbubbles can be identified within the strongly connected components of the input digraph after transforming them into directed acyclic graphs. The algorithm by Sung et al. (IEEE ACM Trans Comput Biol Bioinform 12:770-777, 2015) achieves this task in O ( m l o g ( m ) ) -time. The extraction of superbubbles from the transformed components was later improved to by Brankovic et al. (Theor Comput Sci 609:374-383, 2016) resulting in an overall O ( m + n ) -time algorithm. RESULTS: A re-analysis of the mathematical structure of superbubbles showed that the construction of auxiliary DAGs from the strongly connected components in the work of Sung et al. missed some details that can lead to the reporting of false positive superbubbles. We propose an alternative, even simpler auxiliary graph that solved the problem and retains the linear running time for general digraph. Furthermore, we describe a simpler, space-efficient O ( m + n ) -time algorithm for detecting superbubbles in DAGs that uses only simple data structures. IMPLEMENTATION: We present a reference implementation of the algorithm that accepts many commonly used formats for the input graph and provides convenient access to the improved algorithm. https://github.com/Fabianexe/Superbubble.

Coordinate systems for supergenomes.

BACKGROUND: Genome sequences and genome annotation data have become available at ever increasing rates in response to the rapid progress in sequencing technologies. As a consequence the demand for methods supporting comparative, evolutionary analysis is also growing. In particular, efficient tools to visualize-omics data simultaneously for multiple species are sorely lacking. A first and crucial step in this direction is the construction of a common coordinate system. Since genomes not only differ by rearrangements but also by large insertions, deletions, and duplications, the use of a single reference genome is insufficient, in particular when the number of species becomes large. RESULTS: The computational problem then becomes to determine an order and orientations of optimal local alignments that are as co-linear as possible with all the genome sequences. We first review the most prominent approaches to model the problem formally and then proceed to showing that it can be phrased as a particular variant of the Betweenness Problem. It is NP hard in general. As exact solutions are beyond reach for the problem sizes of practical interest, we introduce a collection of heuristic simplifiers to resolve ordering conflicts. CONCLUSION: Benchmarks on real-life data ranging from bacterial to fly genomes demonstrate the feasibility of computing good common coordinate systems.

Expansion of gene clusters, circular orders, and the shortest Hamiltonian path problem.

Clusters of paralogous genes such as the famous HOX cluster of developmental transcription factors tend to evolve by stepwise duplication of its members, often involving unequal crossing over. Gene conversion and possibly other mechanisms of concerted evolution further obfuscate the phylogenetic relationships. As a consequence, it is very difficult or even impossible to disentangle the detailed history of gene duplications in gene clusters. In this contribution we show that the expansion of gene clusters by unequal crossing over as proposed by Walter Gehring leads to distinctive patterns of genetic distances, namely a subclass of circular split systems. Furthermore, when the gene cluster was left undisturbed by genome rearrangements, the shortest Hamiltonian paths with respect to genetic distances coincide with the genomic order. This observation can be used to detect ancient genomic rearrangements of gene clusters and to distinguish gene clusters whose evolution was dominated by unequal crossing over within genes from those that expanded through other mechanisms.