Multiomics-empowered Deep Phenotyping of Ulcerative Colitis Identifies Biomarker Signatures Reporting Functional Remission States.

INTRODUCTION: Ulcerative colitis [UC] is a chronic disease with rising incidence and unclear aetiology. Deep molecular phenotyping by multiomics analyses may provide novel insights into disease processes and characteristic features of remission states. METHODS: UC pathomechanisms were assessed by proteome profiling of human tissue specimens, obtained from five distinct colon locations for each of the 12 patients included in the study. Systemic disease-associated alterations were evaluated thanks to a cross-sectional setting of mass spectrometry-based multiomics analyses comprising proteins, metabolites, and eicosanoids of plasma obtained from UC patients during acute episodes and upon remission, in comparison with healthy controls. RESULTS: Tissue proteome profiling indicated colitis-associated activation of neutrophils, macrophages, B and T cells, fibroblasts, endothelial cells and platelets, and hypoxic stress, and suggested a general downregulation of mitochondrial proteins accompanying the establishment of apparent wound healing-promoting activities including scar formation. Whereas pro-inflammatory proteins were apparently upregulated by immune cells, the colitis-associated epithelial cells, fibroblasts, endothelial cells, and platelets seemed to predominantly contribute anti-inflammatory and wound healing-promoting proteins. Blood plasma proteomics indicated chronic inflammation and platelet activation, whereas plasma metabolomics identified disease-associated deregulations of gut and gut microbiome-derived metabolites. Upon remission several, but not all, molecular candidate biomarker levels recovered back to normal. CONCLUSION: The findings may indicate that microvascular damage and platelet deregulation hardly resolve upon remission, but apparently persist as disease-associated molecular signatures. This study presents local and systemic molecular alterations integrated in a model for UC pathomechanisms, potentially supporting the assessment of disease and remission states in UC patients.

Highly Cytotoxic Molybdenocenes with Strong Metabolic Effects Inhibit Tumour Growth in Mice.

A series of six highly lipophilic Cp-substituted molybdenocenes bearing different bioactive chelating ligands was synthesized and characterized by NMR spectroscopy, mass spectrometry and X-ray crystallography. In vitro experiments showed a greatly increased cytotoxic potency when compared to the non-Cp-substituted counterparts. In vivo experiments performed with the dichlorido precursor, (Ph2 C-Cp)2 MoCl2 and the in vitro most active complex, containing the thioflavone ligand, showed an inhibition of tumour growth. Proteomic studies on the same two compounds demonstrated a significant regulation of tubulin-associated and mitochondrial inner membrane proteins for both compounds and a strong metabolic effect of the thioflavone containing complex.

Tyk2 is a tumor suppressor in colorectal cancer.

Janus kinase Tyk2 is implicated in cancer immune surveillance, but its role in solid tumors is not well defined. We used Tyk2 knockout mice (Tyk2Δ/Δ) and mice with conditional deletion of Tyk2 in hematopoietic (Tyk2ΔHem) or intestinal epithelial cells (Tyk2ΔIEC) to assess their cell type-specific functions in chemically induced colorectal cancer. All Tyk2-deficient mouse models showed a higher tumor burden after AOM-DSS treatment compared to their corresponding wild-type controls (Tyk2+/+ and Tyk2fl/fl), demonstrating tumor-suppressive functions of Tyk2 in immune cells and epithelial cancer cells. However, specific deletion of Tyk2 in hematopoietic cells or in intestinal epithelial cells was insufficient to accelerate tumor progression, while deletion in both compartments promoted carcinoma formation. RNA-seq and proteomics revealed that tumors of Tyk2Δ/Δ and Tyk2ΔIEC mice were immunoedited in different ways with downregulated and upregulated IFNγ signatures, respectively. Accordingly, the IFNγ-regulated immune checkpoint Ido1 was downregulated in Tyk2Δ/Δ and upregulated in Tyk2ΔIEC tumors, although both showed reduced CD8+ T cell infiltration. These data suggest that Tyk2Δ/Δ tumors are Ido1-independent and poorly immunoedited while Tyk2ΔIEC tumors require Ido1 for immune evasion. Our study shows that Tyk2 prevents Ido1 expression in CRC cells and promotes CRC immune surveillance in the tumor stroma. Both of these Tyk2-dependent mechanisms must work together to prevent CRC progression.

An Anticancer Rhenium Tricarbonyl Targets Fe-S Cluster Biogenesis in Ovarian Cancer Cells.

Target identification remains a critical challenge in inorganic drug discovery to deconvolute potential polypharmacology. Herein, we describe an improved approach to prioritize candidate protein targets based on a combination of dose-dependent chemoproteomics and treatment effects in living cancer cells for the rhenium tricarbonyl compound TRIP. Chemoproteomics revealed 89 distinct dose-dependent targets with concentrations of competitive saturation between 0.1 and 32 µM despite the broad proteotoxic effects of TRIP. Target-response networks revealed two highly probable targets of which the Fe-S cluster biogenesis factor NUBP2 was competitively saturated by free TRIP at nanomolar concentrations. Importantly, TRIP treatment led to a down-regulation of Fe-S cluster containing proteins and upregulated ferritin. Fe-S cluster depletion was further verified by assessing mitochondrial bioenergetics. Consequently, TRIP emerges as a first-in-class modulator of the scaffold protein NUBP2, which disturbs Fe-S cluster biogenesis at sub-cytotoxic concentrations in ovarian cancer cells.

A Proteomic Platform Enables to Test for AML Normalization In Vitro.

Acute promyelocytic leukaemia (APL) can be cured by the co-administration of arsenic trioxide (ATO) and all-trans retinoic acid (ATRA). These small molecules relieve the differentiation blockade of the transformed promyelocytes and trigger their maturation into functional neutrophils, which are physiologically primed for apoptosis. This normalization therapy represents a compelling alternative to cytotoxic anticancer chemotherapy, but lacks an in vitro model system for testing the efficiency of novel combination treatments consisting of inducers of differentiation and metallopharmaceuticals. Here, using proteome profiling we present an experimental framework that enables characterising the differentiation- and metal-specific effects of the combination treatment in a panel of acute myeloid leukaemia (AML) cell lines (HL-60 and U937), including APL (NB4). Differentiation had a substantial impact on the proteome on the order of 10% of the identified proteins and featured classical markers and transcription factors of myeloid differentiation. Additionally, ATO provoked specific cytoprotective effects in the AML cell lines HL-60 and U937. In HL-60, these effects included an integrated stress response (ISR) in conjunction with redox defence, while proteasomal responses and a metabolic rewiring were observed in U937 cells. In contrast, the APL cell line NB4 did not display such adaptions indicating a lack of plasticity to cope with the metal-induced stress, which may explain the clinical success of this combination treatment. Based on the induction of these cytoprotective effects, we proposed a novel metal-based compound to be used for the combination treatment instead of ATO. The organoruthenium drug candidate plecstatin-1 was previously shown to induce reactive oxygen species and an ISR. Indeed, the plecstatin-1 combination was found to affect similar pathways compared to the ATO combination in HL-60 cells and did not lead to cytoprotective response signatures in NB4. Moreover, the monocytic cell line U937 showed a low plasticity to cope with the plecstatin-1 combination, which suggests that this combination might achieve therapeutic benefit beyond APL. We propose that the cytoprotective plasticity of cancer cells might serve as a general proxy to discover novel combination treatments in vitro.

Dichotomous Responses to Chronic Fetal Hypoxia Lead to a Predetermined Aging Phenotype.

Hypoxia-induced intrauterine growth restriction increases the risk for cardiovascular, renal, and other chronic diseases in adults, representing thus a major public health problem. Still, not much is known about the fetal mechanisms that predispose these individuals to disease. Using a previously validated mouse model of fetal hypoxia and bottom-up proteomics, we characterize the response of the fetal kidney to chronic hypoxic stress. Fetal kidneys exhibit a dichotomous response to chronic hypoxia, comprising on the one hand cellular adaptations that promote survival (glycolysis, autophagy, and reduced DNA and protein synthesis), but on the other processes that induce a senescence-like phenotype (infiltration of inflammatory cells, DNA damage, and reduced proliferation). Importantly, chronic hypoxia also reduces the expression of the antiaging proteins klotho and Sirt6, a mechanism that is evolutionary conserved between mice and humans. Taken together, we uncover that predetermined aging during fetal development is a key event in chronic hypoxia, establishing a solid foundation for Barker's hypothesis of fetal programming of adult diseases. This phenotype is associated with a characteristic biomarker profile in tissue and serum samples, exploitable for detecting and targeting accelerated aging in chronic hypoxic human diseases.

Fetal Immunomodulatory Environment Following Cartilage Injury-The Key to CARTILAGE Regeneration?

Fetal cartilage fully regenerates following injury, while in adult mammals cartilage injury leads to osteoarthritis (OA). Thus, in this study, we compared the in vivo injury response of fetal and adult ovine articular cartilage histologically and proteomically to identify key factors of fetal regeneration. In addition, we compared the secretome of fetal ovine mesenchymal stem cells (MSCs) in vitro with injured fetal cartilage to identify potential MSC-derived therapeutic factors. Cartilage injury caused massive cellular changes in the synovial membrane, with macrophages dominating the fetal, and neutrophils the adult, synovial cellular infiltrate. Correspondingly, proteomics revealed differential regulation of pro- and anti-inflammatory mediators and growth-factors between adult and fetal joints. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult compared to fetal cartilage following injury. In contrast, several immunomodulating proteins and growth factors were expressed significantly higher in the fetus than the adult. Comparison of the in vitro MSCs proteome with the in vivo fetal regenerative signature revealed shared upregulation of 17 proteins, suggesting their therapeutic potential. Biomimicry of the fetal paracrine signature to reprogram macrophages and modulate inflammation could be an important future research direction for developing novel therapeutics.

Finger sweat analysis enables short interval metabolic biomonitoring in humans.

Metabolic biomonitoring in humans is typically based on the sampling of blood, plasma or urine. Although established in the clinical routine, these sampling procedures are often associated with a variety of compliance issues, which are impeding time-course studies. Here, we show that the metabolic profiling of the minute amounts of sweat sampled from fingertips addresses this challenge. Sweat sampling from fingertips is non-invasive, robust and can be accomplished repeatedly by untrained personnel. The sweat matrix represents a rich source for metabolic phenotyping. We confirm the feasibility of short interval sampling of sweat from the fingertips in time-course studies involving the consumption of coffee or the ingestion of a caffeine capsule after a fasting interval, in which we successfully monitor all known caffeine metabolites as well as endogenous metabolic responses. Fluctuations in the rate of sweat production are accounted for by mathematical modelling to reveal individual rates of caffeine uptake, metabolism and clearance. To conclude, metabotyping using sweat from fingertips combined with mathematical network modelling shows promise for broad applications in precision medicine by enabling the assessment of dynamic metabolic patterns, which may overcome the limitations of purely compositional biomarkers.

Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function.

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1-10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613.

Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways.

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.

Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8.

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.

Octenidine-based hydrogel shows anti-inflammatory and protease-inhibitory capacities in wounded human skin.

Octenidine dihydrochloride (OCT) is a widely used antiseptic molecule, promoting skin wound healing accompanied with improved scar quality after surgical procedures. However, the mechanisms by which OCT is contributing to tissue regeneration are not yet completely clear. In this study, we have used a superficial wound model by tape stripping of ex vivo human skin. Protein profiles of wounded skin biopsies treated with OCT-containing hydrogel and the released secretome were analyzed using liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. Proteomics analysis of OCT-treated skin wounds revealed significant lower levels of key players in tissue remodeling as well as reepithelization after wounding such as pro-inflammatory cytokines (IL-8, IL-6) and matrix-metalloproteinases (MMP1, MMP2, MMP3, MMP9) when compared to controls. In addition, enzymatic activity of several released MMPs into culture supernatants was significantly lower in OCT-treated samples. Our data give insights on the mode of action based on which OCT positively influences wound healing and identified anti-inflammatory and protease-inhibitory activities of OCT.

Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture.

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.

Inward Outward Signaling in Ovarian Cancer: Morpho-Phospho-Proteomic Profiling Upon Application of Hypoxia and Shear Stress Characterizes the Adaptive Plasticity of OVCAR-3 and SKOV-3 Cells.

With the onset of resistance, ovarian cancer cells display almost unpredictable adaptive potential. This may derive from the tumor genetic ancestry and can be additionally tailored by post translational protein modifications (PTMs). In this study, we took advantage of high-end (phospho)-proteome analysis combined with multiparametric morphometric profiling in high-grade serous (OVCAR-3) and non-serous (SKOV-3) ovarian carcinoma cells. For functional experiments, we applied two different protocols, representing typical conditions of the abdominal cavity and of the growing tumor tissue: on the one side hypoxia (oxygen 1%) which develops within the tumor mass or is experienced during migration/extravasation in non-vascularized areas. On the other hand, fluid shear stress (250 rpm, 2.8 dyn/cm2) which affects tumor surface in the peritoneum or metastases in the bloodstream. After 3 hours incubation, treatment groups were clearly distinguishable by PCA analysis. Whereas basal proteome profiles of OVCAR-3 and SKOV-3 cells appeared almost unchanged, phosphoproteome analysis revealed multiple regulatory events. These affected primarily cellular structure and proliferative potential and consolidated in the proteome signature after 24h treatment. Upon oxygen reduction, metabolism switched toward glycolysis (e.g. upregulation hexokinase-2; HK2) and cell size increased, in concerted regulation of pathways related to Rho-GTPases and/or cytoskeletal elements, resembling a vasculogenic mimicry response. Shear stress regulated proteins governing cell cycle and structure, as well as the lipid metabolism machinery including the delta(14)-sterol reductase, kinesin-like proteins (KIF-22/20A) and the actin-related protein 2/3 complex. Independent microscopy-based validation experiments confirmed cell-type specific morphometric responses. In conclusion, we established a robust workflow enabling the description of the adaptive potential of ovarian cancer cells to physical and chemical stressors typical for the abdominal cavity and supporting the identification of novel molecular mechanisms sustaining tumor plasticity and pharmacologic resistance.

Child murder in the Early Bronze Age: proteomic sex identification of a cold case from Schleinbach, Austria.

The identification of sex-specific peptides in human tooth enamel by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) represents a quantum leap for the study of childhood and social relations more generally. Determining sex-related differences in prehistoric child rearing and mortality has been hampered by the insufficient accuracy in determining the biological sex of juveniles. We conducted mass spectrometric analysis to identify sex-specific peptides in the dental enamel of a child from a settlement pit of the Early Bronze Age settlement of Schleinbach, Austria (c. 1950-1850 bc). Four perimortal impression fractures on the skull of a 5-6-year-old child indicate an intentional killing, with a co-buried loom weight as possible murder weapon. Proteomic analysis, conducted for the first time on prehistoric teeth in Austria, determined the child's sex as male. While we cannot conclusively determine whether the child was the victim of conflicts between village groups or was slain by members of his own community, we suggest that contextual evidence points to the latter. A possible trigger of violence was the follow-on effects of an uncontrolled middle ear infection revealed by an osteological analysis. The boy from Schleinbach highlights the potential for further investigation of gender-biased violence, infanticide and child murder based on the recently developed method of proteomic sex identification.

Structural Similarity with Cholesterol Reveals Crucial Insights into Mechanisms Sustaining the Immunomodulatory Activity of the Mycotoxin Alternariol.

The proliferation of molds in domestic environments can lead to uncontrolled continuous exposure to mycotoxins. Even if not immediately symptomatic, this may result in chronic effects, such as, for instance, immunosuppression or allergenic promotion. Alternariol (AOH) is one of the most abundant mycotoxins produced by Alternariaalternata fungi, proliferating among others in fridges, as well as in humid walls. AOH was previously reported to have immunomodulatory potential. However, molecular mechanisms sustaining this effect remained elusive. In differentiated THP-1 macrophages, AOH hardly altered the secretion of pro-inflammatory mediators when co-incubated with lipopolysaccharide (LPS), opening up the possibility that the immunosuppressive potential of the toxin could be related to an alteration of a downstream pro-inflammatory signaling cascade. Intriguingly, the mycotoxin affected the membrane fluidity in macrophages and it synergistically reacted with the cholesterol binding agent MßCD. In silico modelling revealed the potential of the mycotoxin to intercalate in cholesterol-rich membrane domains, like caveolae, and immunofluorescence showed the modified interplay of caveolin-1 with Toll-like Receptor (TLR) 4. In conclusion, we identified the structural similarity with cholesterol as one of the key determinants of the immunomodulatory potential of AOH.

The Challenge of Classifying Metastatic Cell Properties by Molecular Profiling Exemplified with Cutaneous Melanoma Cells and Their Cerebral Metastasis from Patient Derived Mouse Xenografts.

The prediction of metastatic properties from molecular analyses still poses a major challenge. Here we aimed at the classification of metastasis-related cell properties by proteome profiling making use of cutaneous and brain-metastasizing variants from single melanomas sharing the same genetic ancestry. Previous experiments demonstrated that cultured cells derived from these xenografted variants maintain a stable phenotype associated with a differential metastatic behavior: The brain metastasizing variants produce more spontaneous micro-metastases than the corresponding cutaneous variants. Four corresponding pairs of cutaneous and metastatic cells were obtained from four individual patients, resulting in eight cell-lines presently investigated. Label free proteome profiling revealed significant differences between corresponding pairs of cutaneous and cerebellar metastases from the same patient. Indeed, each brain metastasizing variant expressed several apparently metastasis-associated proteomic alterations as compared with the corresponding cutaneous variant. Among the differentially expressed proteins we identified cell adhesion molecules, immune regulators, epithelial to mesenchymal transition markers, stem cell markers, redox regulators and cytokines. Similar results were observed regarding eicosanoids, considered relevant for metastasis, such as PGE2 and 12-HETE. Multiparametric morphological analysis of cells also revealed no characteristic alterations associated with the cutaneous and brain metastasis variants. However, no correct classification regarding metastatic potential was yet possible with the present data. We thus concluded that molecular profiling is able to classify cells according to known functional categories but is not yet able to predict relevant cell properties emerging from networks consisting of many interconnected molecules. The presently observed broad diversity of molecular patterns, irrespective of restricting to one tumor type and two main classes of metastasis, highlights the important need to develop meta-analysis strategies to predict cell properties from molecular profiling data. Such base knowledge will greatly support future individualized precision medicine approaches.

Cancer-associated fibroblast-derived WNT2 increases tumor angiogenesis in colon cancer.

WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.

Proteome Analysis Reveals Distinct Mitochondrial Functions Linked to Interferon Response Patterns in Activated CD4+ and CD8+ T Cells.

While genetic traits and epigenetic modifications mainly encode cell type-specific effector functions, the eventual outcome is also prone to modulation by post-transcriptional regulation mechanisms. T cells are a powerful model for the investigation of such modulatory effects, as common precursor cells may differentiate either to helper CD4+ T cells or cytotoxic CD8+ cells, which elicit distinct functionalities upon TCR-stimulation. Human primary CD4+ and CD8+ T cells were purified from three individual donors and activated with anti-CD3/CD28 antibodies. Associated proteome alterations were analyzed by high-resolution mass spectrometry using a label-free shotgun approach. Metabolic activation was indicated by upregulation of enzymes related to glycolysis, NADH production, fatty acid synthesis, and uptake as well as amino acid and iron uptake. Besides various inflammatory effector molecules, the mitochondrial proteins CLUH, TFAM, and TOMM34 were found specifically induced in CD4+ T cells. Investigation of overrepresented conserved transcription binding sites by the oPOSSUM software suggested interferon type I inducer IRF1 to cause many of the observed proteome alterations in CD4+ T cells. RT qPCR demonstrated the specific induction of IRF1 in CD4+ T cells only. While the interferon regulatory factor IRF4 was found induced in both T cell subtypes at protein and mRNA level, IRF9 and the type I interferon-induced proteins IFIT1, IFIT3, and MX1 were only found induced in CD4+ T cells. As oxidative stress enhances mitochondrial DNA-dependent type I interferon responses, the present data suggested that mitochondrial activities regulate those cell type-specific signaling pathways. Indeed, we detected mitochondrial superoxide formation predominantly in CD4+ T cells via FACS analysis with MitoSOX™ and confirmed this observation by live cell imaging with confocal microscopy. As interferon signaling regulates important features such as resistance regarding immune checkpoint blockade therapy, the present data may identify potential new targets for the efficient control of highly relevant immune cell properties.

Metabolic, Anti-apoptotic and Immune Evasion Strategies of Primary Human Myeloma Cells Indicate Adaptations to Hypoxia.

Multiple Myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). It develops from a premalignant stage, monoclonal gammopathy of undetermined significance (MGUS), often via an intermediate stage, smoldering MM (SMM). The mechanisms of MM progression have not yet been fully understood, all the more because patients with MGUS and SMM already carry similar initial mutations as found in MM cells. Over the last years, increased importance has been attributed to the tumor microenvironment and its role in the pathophysiology of the disease. Adaptations of MM cells to hypoxic conditions in the BM have been shown to contribute significantly to MM progression, independently from the genetic predispositions of the tumor cells. Searching for consequences of hypoxia-induced adaptations in primary human MM cells, CD138-positive plasma cells freshly isolated from BM of patients with different disease stages, comprising MGUS, SMM, and MM, were analyzed by proteome profiling, which resulted in the identification of 6218 proteins. Results have been made fully accessible via ProteomeXchange with identifier PXD010600. Data previously obtained from normal primary B cells were included for comparative purposes. A principle component analysis revealed three clusters, differentiating B cells as well as MM cells corresponding to less and more advanced disease stages. Comparing these three clusters pointed to the alteration of pathways indicating adaptations to hypoxic stress in MM cells on disease progression. Protein regulations indicating immune evasion strategies of MM cells were determined, supported by immunohistochemical staining, as well as transcription factors involved in MM development and progression. Protein regulatory networks related to metabolic adaptations of the cells became apparent. Results were strengthened by targeted analyses of a selected panel of metabolites in MM cells and MM-associated fibroblasts. Based on our data, new opportunities may arise for developing therapeutic strategies targeting myeloma disease progression.