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Abstract Background The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization. Methods Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions. Results Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus. Conclusions We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.

Abstract Functional and structural alterations of peritubular capillaries (PTCs) are a major determinant of chronic kidney disease (CKD). Using a software-based algorithm for semiautomatic segmentation and morphometric quantification, this study analyzes alterations of PTC shape associated with chronic tubulointerstitial injury in three mouse models and in human biopsies. In normal kidney tissue PTC shape was predominantly elongated, whereas the majority of PTCs associated with chronic tubulointerstitial injury had a rounder shape. This was reflected by significantly reduced PTC luminal area, perimeter and diameters as well as by significantly increased circularity and roundness. These morphological alterations were consistent in all mouse models and human kidney biopsies. The mean circularity of PTCs correlated significantly with categorized glomerular filtration rates and the degree of interstitial fibrosis and tubular atrophy (IFTA) and classified the presence of CKD or IFTA. 3D reconstruction of renal capillaries revealed not only a significant reduction, but more importantly a substantial simplification and reconfiguration of the renal microvasculature in mice with chronic tubulointerstitial injury. Computational modelling predicted that round PTCs can deliver oxygen more homogeneously to the surrounding tissue. Our findings indicate that alterations of PTC shape represent a common and uniform reaction to chronic tubulointerstitial injury independent of the underlying kidney disease.

Osteoclasts are polykaryons formed by cell–cell fusion of highly motile progenitors of the myeloid lineage. Osteoclast activity can preserve skeletal strength and bone homeostasis. However, osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA). Fc receptors activated by IgG immune complexes (IC) can boost osteoclast differentiation and bone loss in the course of RA. In contrast, interferon (IFN) γ secreted by immune cells blocks osteoclast activation. Despite their hypothetical importance in the regulation of osteoclast differentiation in RA, the interconnection between the two pathways has not been described so far. Here, we show by total internal reflection fluorescence (TIRF) microscopy that FcγR3 and IFNγ receptor (IFNγR) locate at close vicinity to each other on the human osteoclast surface. Moreover, the average distance increases during the differentiation process. Interestingly, FcγR and IFNγR activation shapes the position of both receptors to each other. Surprisingly, the inhibitory action of IFNγ on in-vitro human osteoclast differentiation depends on the osteoclast differentiation stage. Indeed, IFNγR activation in early osteoclast precursors completely inhibits the formation of polynucleated osteoclasts, while in premature osteoclasts, it further enhanced their fusion. In addition, gene expression analyses showed that IFNγR activation on early precursor cells but not on premature osteoclasts could induce FcγR expression, suggesting a co-regulation of both receptors on human osteoclast precursors. Phosphokinase array data of precursor cells demonstrate that the observed divergence of IFNγR signaling is dependent on the mitogen−activated protein kinase (MAPK) downstream signaling pathway. Overall, our data indicate that FcγR and IFNγR signaling pathways co-influence the differentiation and activity of osteoclasts dependent on the differentiation state, which might reflect the different stages in RA.

In this work, we analyzed the reliability of alginate-gelatin microcapsules as artificial tumor model. These tumor-like scaffolds are characterized by their composition and stiffness (∼25 kPa), and their capability to restrict -but not hinder- cell migration, proliferation and release from confinement. Hydrogel-based microcapsules were initially utilized to detect differences in mechano-sensitivity between MCF7 and MDA-MB-231 breast cancer cells, and the endothelial cell line EA.hy926. Additionally, we used RNA-seq and transcriptomic methods to determine how the culture strategy (i.e. 2D v/s 3D) may pre-set the expression of genes involved in multidrug resistance, being then validated by performing cytotoxicological tests and assays of cell morphology. Our results show that both breast cancer cells can generate elongated multicellular spheroids inside the microcapsules, prior being released (mimicking intravasation stages), a behavior which was not observed in endothelial cells. Further, we demonstrate that cells isolated from 3D scaffolds show resistance to cisplatin, a process which seems to be strongly influenced by mechanical stress, instead of hypoxia. We finally discuss the role played by aneuploidy in malignancy and resistance to anticancer drugs, based on the increased number of polynucleated cells found within these microcapsules. Overall, our outcomes demonstrate that alginate-gelatin microcapsules represent a simple, yet very accurate tumor-like model, enabling us to mimic the most relevant malignant hints described in vivo , suggesting that confinement and mechanical stress need to be considered when studying pathogenicity and drug resistance of cancer cells in vitro. In this work, we analyzed the reliability of alginate-gelatin microcapsules as an artificial tumor model. These scaffolds are characterized by their composition, elastic properties, and their ability to restrict cell migration, proliferation, and release from confinement. Our results demonstrate four novel outcomes: (i) studying cell migration and proliferation in 3D enabled discrimination between malignant and non-pathogenic cells, (ii) studying the cell morphology of cancer aggregates entrapped in alginate-gelatin microcapsules enabled determination of malignancy degree in vitro, (iii) determination that confinement and mechanical stress, instead of hypoxia, are required to generate clones resistant to anticancer drugs (i.e. cisplatin), and (iv) evidence that resistance to anticancer drugs could be due to the presence of polynucleated cells localized inside polymer-based artificial tumors. [Display omitted] [ABSTRACT FROM AUTHOR]

Advanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored. Spontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines. The conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N -glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N -acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ–resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N -glycosylation, as well as IFNγRα protein stability and signaling. Together, our results demonstrated that tumor-associated changes in N -glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC. [Display omitted] Interferon-γ-receptor protein stability and signaling are impaired in colorectal carcinoma cells through tumor-associated N -glycosylation defects, leading to acquired interferon-γ resistance and poor prognosis. [ABSTRACT FROM AUTHOR]

Background: The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization., Methods: Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions., Results: Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus., Conclusions: We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus., (© 2024. The Author(s).)

Osteoclasts are polykaryons formed by cell-cell fusion of highly motile progenitors of the myeloid lineage. Osteoclast activity can preserve skeletal strength and bone homeostasis. However, osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA). Fc receptors activated by IgG immune complexes (IC) can boost osteoclast differentiation and bone loss in the course of RA. In contrast, interferon (IFN) γ secreted by immune cells blocks osteoclast activation. Despite their hypothetical importance in the regulation of osteoclast differentiation in RA, the interconnection between the two pathways has not been described so far. Here, we show by total internal reflection fluorescence (TIRF) microscopy that FcγR3 and IFNγ receptor (IFNγR) locate at close vicinity to each other on the human osteoclast surface. Moreover, the average distance increases during the differentiation process. Interestingly, FcγR and IFNγR activation shapes the position of both receptors to each other. Surprisingly, the inhibitory action of IFNγ on in-vitro human osteoclast differentiation depends on the osteoclast differentiation stage. Indeed, IFNγR activation in early osteoclast precursors completely inhibits the formation of polynucleated osteoclasts, while in premature osteoclasts, it further enhanced their fusion. In addition, gene expression analyses showed that IFNγR activation on early precursor cells but not on premature osteoclasts could induce FcγR expression, suggesting a co-regulation of both receptors on human osteoclast precursors. Phosphokinase array data of precursor cells demonstrate that the observed divergence of IFNγR signaling is dependent on the mitogen-activated protein kinase (MAPK) downstream signaling pathway. Overall, our data indicate that FcγR and IFNγR signaling pathways co-influence the differentiation and activity of osteoclasts dependent on the differentiation state, which might reflect the different stages in RA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Groetsch, Schachtschabel, Tripal, Schmid, Smith, Schett and Bozec.)

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelial-mesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Summary: Creating 3D animations from microscopy data is computationally expensive and requires high-end hardware. We therefore developed 3Dscript.server, a 3D animation software that runs as a service on dedicated, shared workstations. Using 3Dscript as the underlying rendering engine, it offers unique features not found in existing software: rendering is performed completely server-side. The target animation is specified on the client without the rendering engine, eliminating any hardware requirements client-side. Still, defining an animation is intuitive due to 3Dscript's natural language-based animation description. We implemented a new OMERO web app to utilize 3Dscript.server directly from the OMERO web interface; a Fiji client to use 3Dscript.server from Fiji for integration into image processing pipelines; and batch scripts to run 3Dscript.server on compute clusters for large-scale visualization projects., Availability and Implementation: Source code and documentation is available at https://github.com/bene51/omero_3Dscript, https://github.com/bene51/3Dscript.server and https://github.com/bene51/3Dscript.cluster., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press.)