For patients diagnosed with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) or very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), fasting episodes or high-energy demands remain life threatening. Due to the low incidence, clinical trials for novel LCHADD/VLCADD therapies are limited, and the current mouse models only partially reflect human symptoms. Here, we report the use of mitochondrial morphology and 3D bioprinted, vascularized tissue models to establish a robust testing platform for dietary-based and experimental treatment approaches. Through this platform, we were able to demonstrate, that mitochondrial morphology is strictly regulated by NOX2-driven ROS formation. Treatment of LCHADD/VLCADD-derived fibroblasts with the NOX2-inhibitor naloxone led to the reassembly of mitochondrial structures controlled by DNM1L/MFN2. Using RNA transcriptomics, we identified a pro-fibrotic phenotype in LCHADD and VLCADD patients that impaired vessel formation in fully 3D bioprinted human tissue equivalents. Metabolic supplementation with dietary approaches, which are used in standard therapy, partially improved vascularization. Naloxone induced the strongest improvement, restoring vessel length and network complexity to those of healthy controls, suggesting increased oxidative stress as main driver. Interestingly, naloxone had no effect on healthy fibroblasts, underscoring its safety. Taken together, these results suggest the opioid antagonist naloxone as a potential rescue medication during LCHADD/VLCADD-driven metabolic crises.

Peroxisome proliferator-activated receptor-α (PPARα) is a transcription factor expressed in the skin, where its physiological role remains largely unknown. In this work, we found decreased bacterial density and dysbiosis associated with reduced amounts of Staphylococcus in the skin of PPARα-deficient mice compared to littermate controls. The NOD2 pathway was enhanced, as shown by up-regulation of both NOD2 and its downstream targets, including the antimicrobial peptides mBD3 and mBD4. Moreover, the Th17 immune response was enhanced in keratinocytes and epidermal dendritic T cells. Topical treatment of PPARα-deficient mice with an antibacterial solution did not normalize the upregulation of the innate immunity in keratinocytes. However, the up-regulation of FLG and excessive oxidative stress detected in the epidermis of PPARα-deficient mice were markedly inhibited by topical antibacterial treatment. Thus, our study highlights the overarching role of PPARα in shaping skin immune system and microbiota, with the latter largely controlling FLG expression and inducing oxidative stress in keratinocytes.

Obesity and its associated sequelae have become a major global health concern. While ester-linked lipids are well-established regulators of adipocyte function and energy storage. Emerging evidence from cohort studies suggests that ether-linked lipid levels are also altered during the development of acquired obesity. The genes of two extra-peroxisomal enzymes of ether lipid metabolism, alkylglycerol monooxygenase (AGMO) and plasmanylethanolamine desaturase (PEDS1), have recently been identified, but their physiological roles in humans remain poorly understood. We conducted a monocentric, cross-sectional study analyzing subcutaneous adipose tissue from 30 patients undergoing abdominoplasty. To dissect the function of AGMO and PEDS1, we combined in vitro knockdown experiments in adipocyte-derived stem cells with pulse-chase tracing of a labeled ether lipid precursor. Untargeted lipidomics using liquid chromatography–tandem mass spectrometry was applied to in vivo differentiated adipocytes to assess the impact of AGMO activity on phospholipid composition. To explore a systemic relevance for AGMO and PEDS1 in humans, enzyme activity and gene expression data were correlated with blood lipid and metabolic parameters. We demonstrate that AGMO and PEDS1 are critical regulators of ether lipid subclass balance during adipocyte differentiation. Both enzymes maintained plasmanyl- and plasmenylphospholipid homeostasis without altering total ether lipid levels, indicating dynamic remodeling rather than a metabolic bottleneck. In vivo, AGMO activity reshaped the molecular phospholipid composition of primary adipocytes. Importantly, expression and activity of AGMO and PEDS1 correlated with circulating cholesterol, triglycerides, and lipoprotein particle levels, linking adipose ether lipid metabolism to systemic lipid regulation. This is the first human study highlighting AGMO and PEDS1 as key determinants of adipose ether lipid remodeling with systemic metabolic relevance. By connecting adipocyte ether lipid metabolism to circulating lipid profiles, our findings point to AGMO and PEDS1 as promising candidates for further investigation in the context of obesity and metabolic syndrome, and may warrant further exploration as potential contributors to mitigating metabolic dysregulation.

Due to their pivotal roles in tumor progression and therapy resistance, cancer-associated fibroblasts (CAF) are considered key therapeutic targets with loss of stromal androgen receptor (AR) a poorly understood hallmark of aggressive prostate cancer (PCa). A paucity of pre-clinical models however has hampered functional studies of CAF heterogeneity. We demonstrate that our newly-generated CAF biobank contains three FAP+-fibroblast subtypes, each with unique molecular and functional traits. Cultures with an early-activated phenotype expressed the highest levels of AR and exhibited AR-dependent growth. Consistently, stromal cells expressing early-activation markers co-expressed nuclear AR in clinical specimens and were enriched in pre-neoplastic lesions/low-grade PCa. Conversely, myofibroblastic CAF (myCAF), which expressed low AR levels in vitro and in vivo and were proliferatively-insensitive to AR signaling modulation, constituted the predominant CAF subpopulation in stromogenic high-grade PCa and castration-resistant LACP9 patient-derived xenografts. Exacerbation of the myCAF state upon castration of LAPC9-bearing hosts underscored these findings. Mechanistically, AR loss in myCAF was driven by an NFκB-TGFβ1-YAP1 axis, whose combined targeting synergistically repressed myofibroblastic hallmarks and impaired autophagic flux, effects that were potentiated by enzalutamide resulting in myCAF cell death. Collectively, these findings provide a mechanistic rationale for adjuvant targeting of the YAP1-TGFβ signaling axis to improve patient outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Due to their pivotal roles in tumor progression and therapy resistance, cancer-associated fibroblasts (CAF) are considered key therapeutic targets with loss of stromal androgen receptor (AR) a poorly understood hallmark of aggressive prostate cancer (PCa). A paucity of pre-clinical models however has hampered functional studies of CAF heterogeneity. We demonstrate that our newly generated CAF biobank contains three FAP+-fibroblast subtypes, each with unique molecular and functional traits. Cultures with an early-activated phenotype expressed the highest levels of AR and exhibited AR-dependent growth, whereby AR inhibition suppressed their migration. Consistently, stromal cells expressing early-activation markers co-expressed nuclear AR in clinical specimens and were enriched in pre-neoplastic lesions/low-grade PCa. Conversely, myofibroblastic CAF (myCAF) expressed low AR levels in vitro and in vivo, were insensitive at the proliferative and migratory levels to AR signaling modulation and significantly promoted PCa cell invasion in 3D composite collagen networks. Accordingly, myCAF constituted the predominant CAF subpopulation in stromogenic high-grade PCa and were enriched in aggressive disease states in PCa single cell atlases and castration-resistant LACP9 patient-derived xenografts. Exacerbation of the myCAF state upon castration of LAPC9-bearing hosts underscored these findings. Mechanistically, AR loss in myCAF was driven by an NFκB-TGFβ1-YAP1 axis, whose combined pharmacological or genetic targeting synergistically repressed myofibroblastic hallmarks and impaired autophagic flux, effects that were potentiated by enzalutamide resulting in enhanced myCAF cell death. Collectively, data herein provide a mechanistic rationale for stromal AR loss in aggressive PCa and suggest that adjuvant targeting of the YAP1-TGFβ signaling axis may improve patient outcome.

Cellular senescence, a state of irreversible growth arrest, is implicated in various age-related pathologies, including skin aging. In this study, we investigated the role of CLCA2, a calcium-activated chloride channel accessory protein, in cellular senescence and its implications for skin aging. Utilizing UVB and Nutlin3a-induced senescence models, we observed the upregulation of CLCA2 at both transcriptomic and proteomic levels, suggesting its involvement in senescence pathways. Further analysis revealed that the depletion of CLCA2 led to accelerated senescence onset, characterized by classic senescence markers and a unique secretome profile. In 3D skin equivalent models, SEs constructed with CLCA2 knockdown fibroblasts exhibited features reminiscent of aged skin, underscoring the importance of CLCA2 in maintaining skin homeostasis. Our findings highlight CLCA2 as a novel regulator of cellular senescence and its potential implications for skin aging mechanisms.

Skin aging is the result of two types of aging, "intrinsic aging" an inevitable consequence of physiologic and genetically determined changes and "extrinsic aging," which is dependent on external factors such as exposure to sunlight, smoking, and dietary habits. UVB causes skin injury through the generation of free radicals and other oxidative byproducts, also contributing to DNA damage. Appearance and accumulation of senescent cells in the skin are considered one of the hallmarks of aging in this tissue. Mitochondria play an important role for the development of cellular senescence, in particular stress-induced senescence of human cells. However, many aspects of mitochondrial physiology relevant to cellular senescence and extrinsic skin aging remain to be unraveled. Here, we demonstrate that mitochondria damaged by UVB irradiation of human dermal fibroblasts (HDF) are eliminated by NIX-dependent mitophagy and that this process is important for cell survival under these conditions. Additionally, UVB-irradiation of human dermal fibroblasts (HDF) induces the shedding of extracellular vesicles (EVs), and this process is significantly enhanced in UVB-irradiated NIX-depleted cells. Our findings establish NIX as the main mitophagy receptor in the process of UVB-induced senescence and suggest the release of EVs as an alternative mechanism of mitochondrial quality control in HDF.

Despite significant therapeutic advances in recent years, treatment of metastatic prostate cancer (PCa) remains palliative, owing to the inevitable occurrence of drug resistance. There is increasing evidence that epithelial glucocorticoid receptor (GR) signaling and changes in the tumor-microenvironment (TME) play important roles in this process. Since glucocorticoids (GCs) are used as concomitant medications in the course of PCa treatment, it is essential to investigate the impact of GCs on stromal GR signaling in the TME. Therefore, general GR mRNA and protein expression was assessed in radical prostatectomy specimens and metastatic lesions. Elevated stromal GR signaling after GC treatment resulted in altered GR-target gene, soluble protein expression, and in a morphology change of immortalized and primary isolated cancer-associated fibroblasts (CAFs). Subsequently, these changes affected proliferation, colony formation, and 3D-spheroid growth of multiple epithelial PCa cell models. Altered expression of extra-cellular matrix (ECM) and adhesion-related proteins led to an ECM remodeling. Notably, androgen receptor pathway inhibitor treatments did not affect CAF viability. Our findings demonstrate that GC-mediated elevated GR signaling has a major impact on the CAF secretome and the ECM architecture. GC-treated fibroblasts significantly influence epithelial tumor cell growth and must be considered in future therapeutic strategies.

Screening for PR mRNA (left) and protein (right) expression in multiple breast and prostate cancer cell lines including positive (MCF7, T47D) and negative controls (MDA MB231, BT20).

BackgroundChronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment.MethodsWe examined CD45-/CD31-/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR.ResultsCD45-/CD31-/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs.ConclusionPreadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

Analysis of GR mRNA and protein expression in long-term enzalutamide treated LNCaPabl, DUCaP, LAPC4, and LNCaP cells was performed by qRT-PCR and Western blot, respectively.

E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells.

Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine also known as a mitokine; however, its role in mitochondrial homeostasis and cellular senescence remained elusive. We show here that knocking down GDF15 expression in human dermal fibroblasts induced mitochondrial dysfunction and premature senescence, associated with a distinct senescence-associated secretory phenotype. Fibroblast-specific loss of GDF15 expression in a model of 3D reconstructed human skin induced epidermal thinning, a hallmark of skin aging. Our results suggest GDF15 to play a so far undisclosed role in mitochondrial homeostasis to delay both the onset of cellular senescence and the appearance of age-related changes in a 3D human skin model.

Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel—tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma—tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Despite increasing options for treatment of castration-resistant prostate cancer, development of drug resistance is inevitable. The glucocorticoid receptor (GR) is a prime suspect for acquired therapy resistance, as prostate cancer (PCa) cells are able to increase GR signaling during anti-androgen therapy and thereby circumvent androgen receptor (AR)-blockade and cell death. As standard AR-directed therapies fail to block the GR and GR inhibitors might result in intolerable side effects, the identification of GR signature genes, which are better suited for a targeted approach, is of clinical importance. Therefore, the specific epithelial and stromal GR signature was determined in cancer-associated fibroblasts as well as in abiraterone and enzalutamide-resistant cells after glucocorticoid (GC) treatment. Microarray and ChIP analysis identified MAO-A as a directly up-regulated mutual epithelial and stromal GR target, which is induced after GC treatment and during PCa progression. Elevated MAO-A levels were confirmed in in vitro cell models, in primary tissue cultures after GC treatment, and in patients after neoadjuvant chemotherapy with GCs. MAO-A expression correlates with GR/AR activity as well as with a reduced progression-free survival. Pharmacological MAO-A inhibition combined with 2nd generation AR signaling inhibitors or chemotherapeutics results in impaired growth of androgen-dependent, androgen-independent, and long-term anti-androgen-treated cells. In summary, these findings demonstrate that targeting MAO-A represents an innovative therapeutic strategy to synergistically block GR and AR dependent PCa cell growth and thereby overcome therapy resistance.

Adipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades. However, much less is known about ASC and adipocytes outside classical fat tissue depots and their necessity in tissue remodeling after injury. Therefore, we investigated the etiology of adipocytes in human granulation tissue and define their possible role wound healing. Identification of human wound tissue adipocytes was determined by immunohistochemical staining of granulation tissue sections from patients undergoing surgical debridement. Stromal cell fractions from granulation tissue and subcutaneous fat tissue were generated by collagenase type II-based protocols. Pro- and anti-inflammatory wound bed conditions were mimicked by THP1- and CD14+ monocyte-derived macrophage models in vitro. Effects of macrophage secretome on ASC differentiation and metabolism were determined by immunoblotting, flow cytometry, and microscopy assessing early and late adipocyte differentiation states. Functional rescuing experiments were conducted by lentiviral transduction of wildtype PPARG, IL1RA, and N-chlorotaurine (NCT) treatment. Single and clustered adipocyte populations were detected in 11 out of 13 granulation tissue specimens and single-cell suspensions from granulation tissue showed adipogenic differentiation potential. Pro-inflammatory signaling by IFNG/LPS-stimulated macrophages (M (IFNG/LPS)) inhibited the maturation of lipid droplets in differentiated ASC. In contrast, anti-inflammatory IL4/IL13-activated macrophages (M (IL4/IL13)) revealed minor effects on adipocyte development. The M (IFNG/LPS)-induced phenotype was associated with a switch from endogenous fatty acid synthesis to glycolysis-dominated cell metabolism and increased pro-inflammatory cytokine production. Impaired adipogenesis was associated with increased, but seemingly non-functional, CEBPB levels, which failed to induce downstream PPARG and CEBPA. Neither transgenic PPARG overexpression, nor inhibition of IL1B was sufficient to rescue the anti-adipogenic effects induced by IFNG/LPS-activated macrophages. Instead, macrophage co-treatment during stimulation with NCT, a mild oxidant produced by activated granulocytes present in human wounds in vivo, significantly attenuated the anti-adipogenic effects. In conclusion, the appearance of adipocytes in wound tissue indicates a prevailing anti-inflammatory environment that could be promoted by NCT treatment and may be associated with improved healing outcomes.

p57 is a member of the Cip/Kip family of cell cycle inhibitors which restrict the eukaryotic cell cycle by binding to and inhibiting cyclin/CDK complexes. They are considered as tumor suppressors and inactivating genomic mutations of p57 are associated with human overgrowth disorders. Increasing evidence suggests that p57 controls additional cellular processes beyond cell cycle control such as apoptosis, cell migration or transcription. Here we report that p57 can stimulate AP-1 promotor activity. While transactivation by c-Jun is strongly activated by p57, it did not enhance c-Fos induced transcription. This indicates that c-Jun is the target of p57 in the canonical AP-1 heterodimeric transcription factor. We could detect endogenous p57/c-Jun containing complexes in cells by co-immunoprecipitation. The strong stimulation of c-Jun activity is not the consequence of activating phosphorylation in the transactivation domain (TAD) of c-Jun, but rather due to negative interference with c-Jun repressors and positive interference with c-Jun activators. In contrast to full-length p57, the amino- and carboxy-terminal domains of p57 are insufficient for a significant activation of c-Jun induced transcription. When expressed in presence of full length p57, the p57 C -terminus abrogated and the N -terminus enhanced c-Jun activation. This indicates that the C -terminus may bind and sequester a putative activator of c-Jun, whereas the N -terminus may sequester a c-Jun repressor. Interestingly, the p57 aminoterminus is sufficient for binding to the two c-Jun repressors HDAC1 and HDAC3. These data are consistent with a model of c-Jun activation where p57 is a part of large nuclear remodeling/transcription complexes. p57 might stimulate transcription by inhibiting transcription repressor proteins like HDACs via its N -terminus and/or attracting transcription activators through its C -terminus. These data suggest that in addition to its role as a CDK inhibitor and tumor suppressor, p57 may also exert tumor promoting functions by activation of the proto-oncoprotein c-Jun.

Alterations of the lipid profile of the stratum corneum have an important role in the pathogenesis of atopic dermatitis (AD) because they contribute to epidermal barrier impairment. However, they have not previously been envisioned as a cellular response to altered metabolic requirements in AD epidermis. In this study, we report that the lipid composition in the epidermis of flaky tail, that is, ft/ft mice mimics that of human lesional AD (ADL) epidermis, both showing a shift toward shorter lipid species. The amounts of C24 and C26 free fatty acids and C24 and C26 ceramides-oxidized exclusively in peroxisomes-were reduced in the epidermis of ft/ft mice despite increased lipid synthesis, similar to that seen in human ADL edpidermis. Increased ACOX1 protein and activity in granular keratinocytes of ft/ft epidermis, altered lipid profile in human epidermal equivalents overexpressing ACOX1, and increased ACOX1 immunostaining in skin biopsies from patients with ADL suggest that peroxisomal β-oxidation significantly contributes to lipid signature in ADL epidermis. Moreover, we show that increased anaerobic glycolysis in ft/ft mouse epidermis is essential for keratinocyte proliferation and adenosine triphosphate synthesis but does not contribute to local inflammation. Thus, this work evidenced a metabolic shift toward enhanced peroxisomal β-oxidation and anaerobic glycolysis in ADL epidermis.

Accumulation of senescent cells promotes the development of age-related pathologies and deterioration. In human skin, senescent cells potentially impair structure and function by secreting a mixture of signaling molecules and proteases that influence neighboring cells and degrade extracellular matrix components, such as elastin and collagen. One of the key underlying mechanisms of senescence and extrinsic skin aging is the increase of intracellular reactive oxygen species and resulting oxidative stress. Tert-butyl hydroperoxide (tBHP) is a known inducer of oxidative stress and cellular damage, acting at least in part by depleting the antioxidant glutathione. Here, we provide a detailed characterization of tBHP-induced senescence in human dermal fibroblasts in monolayer culture. In addition, results obtained with more physiological experimental models revealed that tBHP treated 3D reconstructed skin and ex vivo skin developed signs of chronic tissue damage, displaying reduced epidermal thickness and collagen fiber thinning. We, therefore, propose that tBHP treatment can be used as a model to study the effects of extrinsic skin aging, focusing mainly on the influence of environmental pollution.