RNA-Binding Proteins as Novel Effectors in Osteoblasts and Osteoclasts: A Systems Biology Approach to Dissect the Transcriptional Landscape.

Bone health is ensured by the coordinated action of two types of cells-the osteoblasts that build up bone structure and the osteoclasts that resorb the bone. The loss of balance in their action results in pathological conditions such as osteoporosis. Central to this study is a class of RNA-binding proteins (RBPs) that regulates the biogenesis of miRNAs. In turn, miRNAs represent a critical level of regulation of gene expression and thus control multiple cellular and biological processes. The impact of miRNAs on the pathobiology of various multifactorial diseases, including osteoporosis, has been demonstrated. However, the role of RBPs in bone remodeling is yet to be elucidated. The aim of this study is to dissect the transcriptional landscape of genes encoding the compendium of 180 RBPs in bone cells. We developed and applied a multi-modular integrative analysis algorithm. The core methodology is gene expression analysis using the GENEVESTIGATOR platform, which is a database and analysis tool for manually curated and publicly available transcriptomic data sets, and gene network reconstruction using the Ingenuity Pathway Analysis platform. In this work, comparative insights into gene expression patterns of RBPs in osteoblasts and osteoclasts were obtained, resulting in the identification of 24 differentially expressed genes. Furthermore, the regulation patterns upon different treatment conditions revealed 20 genes as being significantly up- or down-regulated. Next, novel gene-gene associations were dissected and gene networks were reconstructed. Additively, a set of osteoblast- and osteoclast-specific gene signatures were identified. The consolidation of data and information gained from each individual analytical module allowed nominating novel promising candidate genes encoding RBPs in osteoblasts and osteoclasts and will significantly enhance the understanding of potential regulatory mechanisms directing intracellular processes in the course of (patho)physiological bone turnover.

Induction of the sphingosine-1-phosphate signaling pathway by TGF-ß1 during Langerhans-type dendritic cell differentiation.

Gene profiling revealed that the S1P signaling pathway is induced by TGF-ß1 during LC commitment of monocytopoietic cells. Constitutive-active TGF-ß1-S1P signaling seems to elevate the activation threshold of LCs and thereby prevent inappropriate and overshooting immune responses to microbial and physicochemical environmental signals. In turn, signals that lead to LC migration may disrupt this pathway via inhibiting S1P bioavailability.

Singularity and Commonality in Response to SARS-CoV-2 in Lung and Colon Cell Models.

The systemic nature of COVID-19 with multiple extrapulmonary manifestations of disease, largely due to the wide tissue expression of SARS-CoV-2 major entry factors, as well as the patient-specific features of COVID-19 pathobiology, determine important directions for basic and translational research. In the current study, we addressed the questions of singularities and commonalities in cellular responses to SARS-CoV-2 and related SARS-CoV on the basis of compendium-wide analysis of publicly available transcriptomic datasets as part of the herein implemented multi-modular UNCOVIDING approach. We focused on cellular models attributed to the epithelial cells of the respiratory system, the Calu-3 cell line, and epithelial cells of the gastrointestinal tract, the Caco-2 cell line, infected with either SARS-CoV-2 or SARS-CoV. Here, we report the outcome of a comparative analysis based on differentially expressed genes in terms of perturbations and diseases, Canonical pathways, and Upstream Regulators. We furthermore performed compendium-wide analysis across more than 19,000 mRNASeq datasets and dissected the condition-specific gene signatures. Information was gained with respect to common and unique cellular responses and molecular events. We identified that in cell lines of colon or lung origin, both viruses show similarities in cellular responses; by contrast, there are cell type-specific regulators that differed for Calu-3 and Caco-2 cells. Among the major findings is the impact of the interferon system for lung Calu-3 cells and novel links to the liver- and lipid-metabolism-associated responses for colon Caco-2 cells as part of the extrapulmonary pathomechanisms in the course of COVID-19. Among differently expressed genes, we specifically dissected the expression pattern of the APOBEC family members and propose APOBEC3G as a promising intrinsic antiviral factor of the host response to SARS-CoV-2. Overall, our study provides gene expression level evidence for the cellular responses attributed to pulmonary and gastrointestinal manifestations of COVID-19.

Human skin dendritic cell fate is differentially regulated by the monocyte identity factor Kruppel-like factor 4 during steady state and inflammation.

BACKGROUND: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency. OBJECTIVE: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood. METHODS: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification. RESULTS: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-ß1, thereby allowing LC differentiation marked by a low cytokine expression profile. CONCLUSION: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BACKGROUND: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells. RESULTS: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background. CONCLUSIONS: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation.

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.

Immunology of Osteoporosis: A Mini-Review.

Osteoporosis is a major cause of fractures and associated morbidity in the aged population. The pathogenesis of osteoporosis is multifactorial; whereas traditional pathophysiological concepts emphasize endocrine mechanisms, it has been recognized that also components of the immune system have a significant impact on bone. Since 2000, when the term 'osteoimmunology' was coined, novel insights into the role of inflammatory cytokines by influencing the fine-tuned balance between bone resorption and bone formation have helped to explain the occurrence of osteoporosis in conjunction with chronic inflammatory reactions. Moreover, the phenomenon of a low-grade, chronic, systemic inflammatory state associated with aging has been defined as 'inflamm-aging' by Claudio Franceschi and has been linked to age-related diseases such as osteoporosis. Given the tight anatomical and physiological coexistence of B cells and the bone-forming units in the bone marrow, a role of B cells in osteoimmunological interactions has long been suspected. Recent findings of B cells as active regulators of the RANK/RANKL/OPG axis, of altered RANKL/OPG production by B cells in HIV-associated bone loss or of a modulated expression of genes linked to B-cell biology in response to estrogen deficiency support this assumption. Furthermore, oxidative stress and the generation of advanced glycation end products have emerged as links between inflammation and bone destruction.

The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon.

BACKGROUND: The calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown. METHODS AND RESULTS: In a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/ß-catenin pathway as measured by a decrease in nuclear translocation of ß-catenin and transcriptional regulation of genes like GSK-3ß and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29(CaSR) cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype. CONCLUSIONS: The results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.

Extracellular matrix remodeling by bone marrow fibroblast-like cells correlates with disease progression in multiple myeloma.

The pathogenesis of multiple myeloma (MM) is regarded as a multistep process, in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. Molecular events characteristic for the transition from MGUS to MM are still poorly defined. We hypothesized that fibroblast-like cells in the tumor microenvironment are critically involved in the pathogenesis of MM. Therefore, we performed a comparative proteome profiling study, analyzing primary human fibroblast-like cells isolated from the bone marrow of MM, of MGUS, as well as of non-neoplastic control patients. Thereby, a group of extracellular matrix (ECM) proteins, ECM receptors, and ECM-modulating enzymes turned out to be progressively up-regulated in MGUS and MM. These proteins include laminin α4, lysyl-hydroxylase 2, prolyl 4-hydroxylase 1, nidogen-2, integrin α5ß5, c-type mannose receptor 2, PAI-1, basigin, and MMP-2, in addition to PDGF-receptor ß and the growth factor periostin, which are likewise involved in ECM activities. Our results indicate that ECM remodeling by fibroblast-like cells may take place already at the level of MGUS and may become even more pronounced in MM. The identified proteins which indicate the stepwise progression from MGUS to MM may offer new tools for therapeutic strategies.

Plasticity of fibroblasts demonstrated by tissue-specific and function-related proteome profiling.

BACKGROUND: Fibroblasts are mesenchymal stromal cells which occur in all tissue types. While their main function is related to ECM production and physical support, they are also important players in wound healing, and have further been recognized to be able to modulate inflammatory processes and support tumor growth. Fibroblasts can display distinct phenotypes, depending on their tissue origin, as well as on their functional state. RESULTS: In order to contribute to the proteomic characterization of fibroblasts, we have isolated primary human fibroblasts from human skin, lung and bone marrow and generated proteome profiles of these cells by LC-MS/MS. Comparative proteome profiling revealed characteristic differences therein, which seemed to be related to the cell's tissue origin. Furthermore, the cells were treated in vitro with the pro-inflammatory cytokine IL-1beta. While all fibroblasts induced the secretion of Interleukins IL-6 and IL-8 and the chemokine GRO-alpha, other inflammation-related proteins were up-regulated in an apparently tissue-dependent manner. Investigating fibroblasts from tumorous tissues of skin, lung and bone marrow with respect to such inflammation-related proteins revealed hardly any conformity but rather individual and tumor type-related variations. However, apparent up-regulation of IGF-II, PAI-1 and PLOD2 was observed in melanoma-, lung adenocarcinoma- and multiple myeloma-associated fibroblasts, as well as in hepatocellular carcinoma-associated fibroblasts. CONCLUSIONS: Inflammation-related proteome alterations of primary human fibroblasts were determined by the analysis of IL-1beta treated cells. Tumor-associated fibroblasts from different tissue types hardly showed signs of acute inflammation but displayed characteristic functional aberrations potentially related to chronic inflammation. The present data suggest that the state of the tumor microenvironment is relevant for tumor progression and targeted treatment of tumor-associated fibroblasts may support anti-cancer strategies.

Determination of cell type-specific proteome signatures of primary human leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts and melanocytes by comparative proteome profiling.

Cells gain their functional specialization by different protein synthesis. A lot of knowledge with respect to cell type-specific proteins has been collected during the last thirty years. This knowledge was built mainly by using antibodies. Nowadays, modern MS, which supports comprehensive proteome analyses of biological samples, may render possible the search for cell type-specific proteins as well. However, a therefore necessary systematic MS study comprising many different cell types has not been performed until now. Here we present a proteome analysis strategy supporting the automated and meaningful comparison of any biological samples. We have presently applied this strategy to six different primary human cell types, namely leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts, and melanocytes. Comparative analysis of the resulting proteome profiles allowed us to select proteins specifically identified in one of the six cell types and not in any of the five others. Based on these results, we designated cell type-specific proteome signatures consisting each of six such characteristic proteins. These signatures independently reproduced well-known marker proteins already established for FACS analyses in addition to novel candidate marker proteins. We applied these signatures for the interpretation of proteome profiles obtained from the analyses of hepatocellular carcinoma-associated tissue homogenates and normal liver tissue homogenates. The identification of members of the above described signatures gave us an indication of the presence of characteristic cells in the diseased tissues and thus supported the interpretation of the proteomics data of these complex biological samples.

Microenvironmental and cell intrinsic factors governing human cDC2 differentiation and monocyte reprogramming.

cDC2s occur abundantly in peripheral tissues and arise from circulating blood cDC2s. However, the factors governing cDC2 differentiation in tissues, especially under inflammatory conditions, remained poorly defined. We here found that psoriatic cDC2s express the efferocytosis receptor Axl and exhibit a bone morphogenetic protein (BMP) and p38MAPK signaling signature. BMP7, strongly expressed within the lesional psoriatic epidermis, cooperates with canonical TGF-ß1 signaling for inducing Axl+cDC2s from blood cDC2s in vitro. Moreover, downstream induced p38MAPK promotes Axl+cDC2s at the expense of Axl+CD207+ Langerhans cell differentiation from blood cDC2s. BMP7 supplementation allowed to model cDC2 generation and their further differentiation into LCs from CD34+ hematopoietic progenitor cells in defined serum-free medium. Additionally, p38MAPK promoted the generation of another cDC2 subset lacking Axl but expressing the non-classical NFkB transcription factor RelB in vitro. Such RelB+cDC2s occurred predominantly at dermal sites in the inflamed skin. Finally, we found that cDC2s can be induced to acquire high levels of the monocyte lineage identity factor kruppel-like-factor-4 (KLF4) along with monocyte-derived DC and macrophage phenotypic characteristics in vitro. In conclusion, inflammatory and psoriatic epidermal signals instruct blood cDC2s to acquire phenotypic characteristics of several tissue-resident cell subsets.

Activation-induced cytidine deaminase (AID) linking immunity, chronic inflammation, and cancer.

Activation-induced cytidine deaminase (AID) is critically involved in class switch recombination and somatic hypermutation of Ig loci resulting in diversification of antibodies repertoire and production of high-affinity antibodies and as such represents a physiological tool to introduce DNA alterations. These processes take place within germinal centers of secondary lymphoid organs. Under physiological conditions, AID is expressed predominantly in activated B lymphocytes. Because of the mutagenic and recombinogenic potential of AID, its expression and activity is tightly regulated on different levels to minimize the risk of unwanted DNA damage. However, chronic inflammation and, probably, combination of other not-yet-identified factors are able to create a microenvironment sufficient for triggering an aberrant AID expression in B cells and, importantly, in non-B-cell background. Under these circumstances, AID may target also non-Ig genes, including cancer-related genes as oncogenes, tumor suppressor genes, and genomic stability genes, and modulate both genetic and epigenetic information. Despite ongoing progress, the complete understanding of fundamental aspects is still lacking as (1) what are the crucial factors triggering an aberrant AID expression/activity including the impact of Th2-driven inflammation and (2) to what extent may aberrant AID in human non-B cells lead to abnormal cell state associated with an increased rate of genomic alterations as point mutations, small insertions or deletions, and/or recurrent chromosomal translocations during solid tumor development and progression.

Yugra State University Biological Collection (Khanty-Mansiysk, Russia): general and digitisation overview.

BACKGROUND: The history of biological collections and digitisation initiatives in northern West Siberia is relatively new due to recent development of the region. The Center for Biodiversity Data Mobilization was established to promote the initiative, led by the Yugra State University. This organisation itself has a relatively young collection of biological specimens, which was, until recently, in a disintegrated state and only partly mobilised. The Yugra State University Biological Collection (YSU BC) currently includes three subdivisions differring by history and taxonomic groups, but also by details of management and storage conditions: the Fungarium, the Bryological collection and the Herbarium collection of YSU.The paper describes the general structure of the Yugra State University Biological Collection, its history, storage conditions, management practices, geographical, temporal and taxonomical coverage. The paper is underlined by three datasets of the collections databases published in GBIF, which are described in detail. The databases are managed in Specify 6 and 7 software and accessed through Specify Web Portal and through GBIF. NEW INFORMATION: The Yugra State University Biological Collection made an active reorganisation of physical storage conditions and data management recently, providing the model for other collections in the region. This paper describes the history, general structure, management practices and data management of all three parts of this collection for the first time.Although one part of the collection (Fungarium YSU) was mobilised earlier, last year, we mobilised data of the Bryological and Vascular plants (Herbarium) collections. The three datasets of the corresponding collections in GBIF were increased by about 6000 georeferenced records during the last year.

Elimination of LRVs Elicits Different Responses in Leishmania spp.

Leishmaniaviruses (LRVs) have been demonstrated to enhance progression of leishmaniasis, a vector-transmitted disease with a wide range of clinical manifestations that is caused by flagellates of the genus Leishmania. Here, we used two previously proposed strategies of the LRV ablation to shed light on the relationships of two Leishmania spp. with their respective viral species (L. guyanensis, LRV1 and L. major, LRV2) and demonstrated considerable difference between two studied systems. LRV1 could be easily eliminated by the expression of exogenous capsids regardless of their origin (the same or distantly related LRV1 strains, or even LRV2), while LRV2 was only partially depleted in the case of the native capsid overexpression. The striking differences were also observed in the effects of complete viral elimination with 2'C-methyladenosine (2-CMA) on the transcriptional profiles of these two Leishmania spp. While virtually no differentially expressed genes were detected after the LRV1 removal from L. guyanensis, the response of L. major after ablation of LRV2 involved 87 genes, the analysis of which suggested a considerable stress experienced even after several passages following the treatment. This effect on L. major was also reflected in a significant decrease of the proliferation rate, not documented in L. guyanensis and naturally virus-free strain of L. major. Our findings suggest that integration of L. major with LRV2 is deeper compared with that of L. guyanensis with LRV1. We presume this determines different effects of the viral presence on the Leishmania spp. infections. IMPORTANCE Leishmania spp. represent human pathogens that cause leishmaniasis, a widespread parasitic disease with mild to fatal clinical manifestations. Some strains of leishmaniae bear leishmaniaviruses (LRVs), and this has been shown to aggravate disease course. We investigated the relationships of two distally related Leishmania spp. with their respective LRVs using different strategies of virus removal. Our results suggest the South American L. guyanensis easily loses its virus with no important consequences for the parasite in the laboratory culture. Conversely, the Old-World L. major is refractory to virus removal and experiences a prominent stress if this removal is nonetheless completed. The drastically different levels of integration between the studied Leishmania spp. and their viruses suggest distinct effects of the viral presence on infections in these species of parasites.

AID and APOBECs as Multifaceted Intrinsic Virus-Restricting Factors: Emerging Concepts in the Light of COVID-19.

The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer.

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC.

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC50 value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.

Interrelations of Sphingolipid and Lysophosphatidate Signaling with Immune System in Ovarian Cancer.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with CD68, LPAR3, SMPD1, PPAP2B, and SMPD2 emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.

Resveratrol Inhibits Key Steps of Steroid Metabolism in a Human Estrogen-Receptor Positive Breast Cancer Model: Impact on Cellular Proliferation.

The role of resveratrol (RES) in preventing breast cancer is controversial, as low concentrations may stimulate the proliferation of estrogen-receptor alpha positive (ERα+) breast cancer cells. As metabolism is the key factor in altering cellular estrogens, thereby influencing breast tumor growth, we investigated the effects of RES on the formation of estrogen metabolites, namely 4-androstene-3,17-dione (AD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone-3-O-sulfate (DHEA-S), estrone (E1), estrone-3-sulfate (E1-S), 17ß-estradiol (E2), 17ß-estradiol-3-O-(ß-D-glucuronide) (E2-G), 17ß-estradiol-3-O-sulfate (E2-S), 16α-hydroxy-17ß-estradiol (estriol, E3), and testosterone (T) in ERα- MDA-MB-231 and ERα+ MCF-7 cells. Incubation of both of the cell lines with the hormone precursors DHEA and E1 revealed that sulfation and glucuronidation were preferred metabolic pathways for DHEA, E1 and E2 in MCF-7 cells, compared with in MDA-MB-231 cells, as the Vmax values were significantly higher (DHEA-S: 2873.0 ± 327.4 fmol/106 cells/h, E1-S: 30.4 ± 2.5 fmol/106 cells/h, E2-S: 24.7 ± 4.9 fmol/106 cells/h, E2-G: 7.29 ± 1.36 fmol/106 cells/h). RES therefore significantly inhibited DHEA-S, E1-S, E2-S and E2-G formation in MCF-7, but not in MDA-MB-231 cells (Kis: E2-S, 0.73 ± 0.07 µM < E1-S, 0.94 ± 0.03 µM < E2-G, 7.92 ± 0.24 µM < DHEA-S, 13.2 ± 0.2 µM). Suppression of these metabolites subsequently revealed twofold higher levels of active E2, concomitant with an almost twofold increase in MCF-7 cell proliferation, which was the most pronounced upon the addition of 5 µM RES. As the content of RES in food is relatively low, an increased risk of breast cancer progression in women is likely to only be observed following the continuous consumption of high-dose RES supplements. Further long-term human studies simultaneously monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the efficacy and safety of RES supplementation, particularly in patients diagnosed with ERα+ breast cancer.