Cells

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18 pages, 2911 KiB

Open AccessEditor’s ChoiceArticle

Flow Cytometric Assessment of FcγRIIIa-V158F Polymorphisms and NK Cell Mediated ADCC Revealed Reduced NK Cell Functionality in Colorectal Cancer Patients

by Phillip Schiele, Stefan Kolling, Stanislav Rosnev, Charlotte Junkuhn, Anna Luzie Walter, Jobst Christian von Einem, Sebastian Stintzing, Wenzel Schöning, Igor Maximilian Sauer, Dominik Paul Modest, Kathrin Heinrich, Lena Weiss, Volker Heinemann, Lars Bullinger, Marco Frentsch and Il-Kang Na

Cells 2025, 14(1), 32; https://doi.org/10.3390/cells14010032 - 31 Dec 2024

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Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells is a key mechanism in anti-cancer therapies with monoclonal antibodies, including cetuximab (EGFR-targeting) and avelumab (PDL1-targeting). Fc gamma receptor IIIa (FcγRIIIa) polymorphisms impact ADCC, yet their clinical relevance in NK cell functionality remains debated. We developed [...] Read more.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells is a key mechanism in anti-cancer therapies with monoclonal antibodies, including cetuximab (EGFR-targeting) and avelumab (PDL1-targeting). Fc gamma receptor IIIa (FcγRIIIa) polymorphisms impact ADCC, yet their clinical relevance in NK cell functionality remains debated. We developed two complementary flow cytometry assays: one to predict the FcγRIIIa-V158F polymorphism using a machine learning model, and a 15-color flow cytometry panel to assess antibody-induced NK cell functionality and cancer-immune cell interactions. Samples were collected from healthy donors and metastatic colorectal cancer (mCRC) patients from the FIRE-6-Avelumab phase II study. The machine learning model accurately predicted the FcγRIIIa-V158F polymorphism in 94% of samples. FF homozygous patients showed diminished cetuximab-mediated ADCC compared to VF or VV carriers. In mCRC patients, NK cell dysfunctions were evident as impaired ADCC, decreased CD16 downregulation, and reduced CD137/CD107a induction. Elevated PD1+ NK cell levels, reduced lysis of PDL1-expressing CRC cells and improved NK cell activation in combination with the PDL1-targeting avelumab indicate that the PD1-PDL1 axis contributes to impaired cetuximab-induced NK cell function. Together, these optimized assays effectively identify NK cell dysfunctions in mCRC patients and offer potential for broader application in evaluating NK cell functionality across cancers and therapeutic settings. Full article

(This article belongs to the Special Issue Advances in the Study of Natural Killer (NK) Cells)

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17 pages, 2689 KiB

Open AccessEditor’s ChoiceArticle

Ezrin Polarization as a Diagnostic Marker for Circulating Tumor Cells in Hepatocellular Carcinoma

by Ibrahim Büdeyri, Olaf Guckelberger, Elsie Oppermann, Dhruvajyoti Roy, Svenja Sliwinski, Felix Becker, Benjamin Struecker, Thomas J. Vogl, Andreas Pascher, Wolf O. Bechstein, Anna Lorentzen, Mathias Heikenwalder and Mazen A. Juratli

Cells 2025, 14(1), 6; https://doi.org/10.3390/cells14010006 - 25 Dec 2024

Viewed by 1362

Abstract

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide, with no precise method for early detection. Circulating tumor cells (CTCs) expressing the dynamic polarity of the cytoskeletal membrane protein, ezrin, have been proposed to [...] Read more.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide, with no precise method for early detection. Circulating tumor cells (CTCs) expressing the dynamic polarity of the cytoskeletal membrane protein, ezrin, have been proposed to play a crucial role in tumor progression and metastasis. This study investigated the diagnostic and prognostic potential of polarized circulating tumor cells (p-CTCs) in HCC patients. CTCs were isolated from the peripheral blood of 20 HCC patients and 18 patients with nonmalignant liver disease (NMLD) via an OncoQuick^® kit and immunostained with Ezrin-Alexa Fluor 488^®, CD146-PE, and CD45-APC. A fluorescence microscopy was then performed for analysis. The HCC group exhibited significantly higher levels of p-CTCs, with median values of 0.56 p-CTCs/mL, compared to 0.02 p-CTCs/mL (p = 0.03) in the NMLD group. CTCs were detected in 95% of the HCC patients, with a sensitivity of 95% and specificity of 89%. p-CTCs were present in 75% of the HCC patients, with a sensitivity of 75% and a specificity of 94%. Higher p-CTC counts were associated with the significantly longer overall survival in HCC patients (p = 0.05). These findings suggest that p-CTCs could serve as valuable diagnostic and prognostic markers for HCC. The incorporation of p-CTCs into diagnostic strategies could enhance therapeutic decision-making and improve patient outcomes. Full article

(This article belongs to the Collection Current Understanding in Cancer Treatment and Monitoring Through Identification of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles)

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17 pages, 2859 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

The Tumor Metabolite 5′-Deoxy-5′Methylthioadenosine (MTA) Inhibits Maturation and T Cell-Stimulating Capacity of Dendritic Cells

by Christina Brummer, Katrin Singer, Frederik Henrich, Katrin Peter, Carolin Strobl, Bernadette Neueder, Christina Bruss, Kathrin Renner, Tobias Pukrop, Wolfgang Herr, Michael Aigner and Marina Kreutz

Cells 2024, 13(24), 2114; https://doi.org/10.3390/cells13242114 - 20 Dec 2024

Viewed by 1365

Abstract

Metabolite accumulation in the tumor microenvironment fosters immune evasion and limits the efficiency of immunotherapeutic approaches. Methylthioadenosine phosphorylase (MTAP), which catalyzes the degradation of 5′-deoxy-5′methylthioadenosine (MTA), is downregulated in many cancer entities. Consequently, MTA accumulates in the microenvironment of MTAP-deficient tumors, where it [...] Read more.

Metabolite accumulation in the tumor microenvironment fosters immune evasion and limits the efficiency of immunotherapeutic approaches. Methylthioadenosine phosphorylase (MTAP), which catalyzes the degradation of 5′-deoxy-5′methylthioadenosine (MTA), is downregulated in many cancer entities. Consequently, MTA accumulates in the microenvironment of MTAP-deficient tumors, where it is known to inhibit tumor-infiltrating T cells and NK cells. However, the impact of MTA on other intra-tumoral immune cells has not yet been fully elucidated. To study the effects of MTA on dendritic cells (DCs), human monocytes were maturated into DCs with (MTA-DC) or without MTA (co-DC) and analyzed for activation, differentiation, and T cell-stimulating capacity. MTA altered the cytokine secretion profile of monocytes and impaired their maturation into dendritic cells. MTA-DCs produced less IL-12 and showed a more immature-like phenotype characterized by decreased expression of the co-stimulatory molecules CD80, CD83, and CD86 and increased expression of the monocyte markers CD14 and CD16. Consequently, MTA reduced the capability of DCs to stimulate T cells. Mechanistically, the MTA-induced effects on monocytes and DCs were mediated by a mechanism beyond adenosine receptor signaling. These results provide new insights into how altered polyamine metabolism impairs the maturation of monocyte-derived DCs and impacts the crosstalk between T and dendritic cells. Full article

(This article belongs to the Collection Novel Advances in Cancer Immunology: From Molecular Mechanisms to Therapeutic Applications)

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20 pages, 3994 KiB

Open AccessEditor’s ChoiceArticle

Discovery of Carbonic Anhydrase 9 as a Novel CLEC2 Ligand in a Cellular Interactome Screen

by Sebastian Hoffmann, Benedict-Tilman Berger, Liane Rosalie Lucas, Felix Schiele and John Edward Park

Cells 2024, 13(24), 2083; https://doi.org/10.3390/cells13242083 - 17 Dec 2024

Viewed by 1441

Abstract

Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently [...] Read more.

Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14^high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin’s lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies. Full article

(This article belongs to the Section Cellular Immunology)

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15 pages, 3500 KiB

Open AccessEditor’s ChoiceArticle

α-Synuclein Deletion Impairs Platelet Function: A Role for SNARE Complex Assembly

by Christopher Sennett, Wanzhu Jia, Jawad S. Khalil, Matthew S. Hindle, Charlie Coupland, Simon D. J. Calaminus, Julian D. Langer, Sean Frost, Khalid M. Naseem, Francisco Rivero, Natalia Ninkina, Vladimir Buchman and Ahmed Aburima

Cells 2024, 13(24), 2089; https://doi.org/10.3390/cells13242089 - 17 Dec 2024

Cited by 1 | Viewed by 1403

Abstract

Granule secretion is an essential platelet function that contributes not only to haemostasis but also to wound healing, inflammation, and atherosclerosis. Granule secretion from platelets is facilitated, at least in part, by Soluble N-ethylmaleimide-Sensitive Factor (NSF) Attachment Protein Receptor (SNARE) complex-mediated granule fusion. [...] Read more.

Granule secretion is an essential platelet function that contributes not only to haemostasis but also to wound healing, inflammation, and atherosclerosis. Granule secretion from platelets is facilitated, at least in part, by Soluble N-ethylmaleimide-Sensitive Factor (NSF) Attachment Protein Receptor (SNARE) complex-mediated granule fusion. Although α-synuclein is a protein known to modulate the assembly of the SNARE complex in other cells, its role in platelet function remains poorly understood. In this study, we provide evidence that α-synuclein is critical for haemostasis using α-synuclein-deficient (^−/−) mice. The genetic deletion of α-synuclein resulted in impaired platelet aggregation, secretion, and adhesion in vitro. In vivo haemostasis models showed that α-synuclein^−/− mice had prolonged bleeding times and activated partial thromboplastin times (aPTTs). Mechanistically, platelet activation induced α-synuclein serine (ser) 129 phosphorylation and re-localisation to the platelet membrane, accompanied by an increased association with VAMP 8, syntaxin 4, and syntaxin 11. This phosphorylation was calcium (Ca²⁺)- and RhoA/ROCK-dependent and was inhibited by prostacyclin (PGI₂). Our data suggest that α-synuclein regulates platelet secretion by facilitating SNARE complex formation. Full article

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16 pages, 4686 KiB

Open AccessEditor’s ChoiceArticle

Subset of DN Memory B Cells Expressing Low Levels of Inhibitory Receptor BTLA Is Enriched in SLE Patients

by Lucie Aubergeon, Renaud Felten, Jacques-Eric Gottenberg, Hélène Dumortier and Fanny Monneaux

Cells 2024, 13(24), 2063; https://doi.org/10.3390/cells13242063 - 13 Dec 2024

Cited by 2 | Viewed by 1386

Abstract

The dialogue between T and B cells can be regulated by different mechanisms, such as co-inhibitory receptors, which therefore play a crucial role in preventing autoimmune diseases such as systemic lupus erythematosus (SLE). B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor [...] Read more.

The dialogue between T and B cells can be regulated by different mechanisms, such as co-inhibitory receptors, which therefore play a crucial role in preventing autoimmune diseases such as systemic lupus erythematosus (SLE). B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor expressed on many myeloid and lymphoid cells. Although peripheral B cells express a very high amount of BTLA, previous works in the context of autoimmunity mainly focused on T cells, and whether BTLA expression on B cells plays a role in the lupus pathogenesis is still unclear. In the present study, we examine the expression of BTLA, as well as its ligand HVEM (Herpesvirus Entry Mediator), on various B cell subsets in lupus patients compared to healthy controls (HCs). We evidenced the existence of double-negative (DN; IgD⁻CD27⁻) memory B cells expressing very low levels of BTLA, which are enhanced in active lupus patients. An in-depth analysis revealed that these BTLA^low DN cells mainly correspond to the newly reported DN3 B cell subset, originally described in the context of SARS-CoV2 infection. These cells display an activated and antibody-secreting cell phenotype, and we propose that their low BTLA expression may favor their expansion and rapid differentiation into plasmablasts in lupus patients. Full article

(This article belongs to the Special Issue Immune Functions and Therapeutic Potential of B Lymphocytes in Autoimmune Disorders)

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17 pages, 10124 KiB

Open AccessEditor’s ChoiceArticle

KSRP Deficiency Attenuates the Course of Pulmonary Aspergillosis and Is Associated with the Elevated Pathogen-Killing Activity of Innate Myeloid Immune Cells

by Vanessa Bolduan, Kim-Alicia Palzer, Frederic Ries, Nora Busch, Andrea Pautz and Matthias Bros

Cells 2024, 13(24), 2040; https://doi.org/10.3390/cells13242040 - 10 Dec 2024

Cited by 1 | Viewed by 998

Abstract

The mRNA-binding protein KSRP (KH-type splicing regulatory protein) is known to modulate immune cell functions post-transcriptionally, e.g., by reducing the mRNA stability of cytokines. It is known that KSRP binds the AU-rich motifs (ARE) that are often located in the 3′-untranslated part of [...] Read more.

The mRNA-binding protein KSRP (KH-type splicing regulatory protein) is known to modulate immune cell functions post-transcriptionally, e.g., by reducing the mRNA stability of cytokines. It is known that KSRP binds the AU-rich motifs (ARE) that are often located in the 3′-untranslated part of mRNA species, encoding dynamically regulated proteins as, for example, cytokines. Innate myeloid immune cells, such as polymorphonuclear neutrophils (PMNs) and macrophages (MACs), eliminate pathogens by multiple mechanisms, including phagocytosis and the secretion of chemo- and cytokines. Here, we investigated the role of KSRP in the phenotype and functions of both innate immune cell types in the mouse model of invasive pulmonary aspergillosis (IPA). Here, KSRP^−/− mice showed lower levels of Aspergillus fumigatus conidia (AFC) and an increase in the frequencies of PMNs and MACs in the lungs. Our results showed that PMNs and MACs from KSRP^−/− mice exhibited an enhanced phagocytic uptake of AFC, accompanied by increased ROS production in PMNs upon stimulation. A comparison of RNA sequencing data revealed that 64 genes related to inflammatory and immune responses were shared between PMNs and MACs. The majority of genes upregulated in PMNs were involved in metabolic processes, cell cycles, and DNA repair. Similarly, KSRP-deficient PMNs displayed reduced levels of apoptosis. In conclusion, our results indicate that KSRP serves as a critical negative regulator of PMN and MAC anti-pathogen activity. Full article

(This article belongs to the Special Issue Innate Immunity in Health and Disease)

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25 pages, 6935 KiB

Open AccessEditor’s ChoiceArticle

Clinical Potential of Novel Microbial Therapeutic LP51 Based on Xerosis-Microbiome Index

by Sukyung Kim, Md Abdur Rahim, Hanieh Tajdozian, Indrajeet Barman, Hyun-A Park, Youjin Yoon, Sujin Jo, Soyeon Lee, Md Sarower Hossen Shuvo, Sung Hae Bae, Hyunji Lee, Sehee Ju, Chae-eun Park, Ho-Kyoung Kim, Jeung Hi Han, Ji-Woong Kim, Sung geon Yoon, Jae Hong Kim, Yang Gyu Choi, Saebim Lee, Hoonhee Seo and Ho-Yeon Song Show full author list Hide full author list

Cells 2024, 13(23), 2029; https://doi.org/10.3390/cells13232029 - 9 Dec 2024

Viewed by 1521

Abstract

Xerosis, characterized by dry, rough skin, causes discomfort and aesthetic concerns, necessitating effective treatment. Traditional treatments often show limited efficacy, prompting the need for innovative therapies. This study highlights the efficacy of microbiome therapeutic LP51, derived from a healthy vaginal microbiome, in improving [...] Read more.

Xerosis, characterized by dry, rough skin, causes discomfort and aesthetic concerns, necessitating effective treatment. Traditional treatments often show limited efficacy, prompting the need for innovative therapies. This study highlights the efficacy of microbiome therapeutic LP51, derived from a healthy vaginal microbiome, in improving xerosis. A double-blind clinical trial involving 43 subjects with dry inner arm skin compared the effects of a 2.9% LP51 extract formulation to a placebo over 4 weeks. The LP51 group exhibited a significant increase in stratum corneum hydration (10.0 A.U.) compared to the placebo group (4.8 A.U.) and a 21.4% decrease in transepidermal water loss (TEWL), whereas the placebo group showed no significant change. LP51 also demonstrated benefits in enhancing skin hydration, improving the skin barrier, and exhibited anti-atopic, anti-inflammatory, and antioxidant properties. Safety was confirmed through in vitro cytotoxicity tests. These effects are attributed to the microbiome-safe component in LP51 and its role in improving xerosis, reflected by an increase in the xerosis-microbiome index, defined by the Firmicutes/Actinobacteria ratio. These findings position microbiome therapeutic LP51 as a promising novel treatment for xerosis. Full article

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17 pages, 5212 KiB

Open AccessEditor’s ChoiceArticle

LINC01270 Regulates the NF-κB-Mediated Pro-Inflammatory Response via the miR-326/LDOC1 Axis in THP-1 Cells

by Imene Arab, Su-Geun Lim, Kyoungho Suk and Won-Ha Lee

Cells 2024, 13(23), 2027; https://doi.org/10.3390/cells13232027 - 8 Dec 2024

Viewed by 1387

Abstract

Long intergenic noncoding (LINC)01270 is a 2278 bp transcript belonging to the intergenic subset of long noncoding (lnc)RNAs. Despite increased reports of LINC01270’s involvement in different diseases, evident research on its effects on inflammation is yet to be achieved. In the present study, [...] Read more.

Long intergenic noncoding (LINC)01270 is a 2278 bp transcript belonging to the intergenic subset of long noncoding (lnc)RNAs. Despite increased reports of LINC01270’s involvement in different diseases, evident research on its effects on inflammation is yet to be achieved. In the present study, we investigated the potential role of LINC01270 in modulating the inflammatory response in the human monocytic leukemia cell line THP-1. Lipopolysaccharide treatment upregulated LINC01270 expression, and siRNA-mediated suppression of LINC01270 enhanced NF-κB activity and the subsequent production of cytokines IL-6, IL-8, and MCP-1. Interestingly, the knockdown of LINC01270 downregulated expression of leucine zipper downregulated in cancer 1 (LDOC1), a novel NF-κB suppressor. An analysis of the LINC01270/micro-RNA (miRNA)/protein interactome profile identified miR-326 as a possible mediator. Synthetic RNA agents that perturb the interaction among LINC01270, miR-326, and LDOC1 mRNA mitigated the changes caused by LINC01270 knockdown in THP-1 cells. Additionally, a luciferase reporter assay in HEK293 cells further confirmed that LINC01270 knockdown enhances NF-κB activation, while its overexpression has the opposite effect. This study provides insight into LINC01270’s role in modulating inflammatory responses to lipopolysaccharide stimulation in THP-1 cells via the miR-326/LDOC1 axis, which negatively regulates NF-κB activation. Full article

(This article belongs to the Special Issue Macrophage Activation and Regulation)

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16 pages, 1709 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Differential Infiltration of Key Immune T-Cell Populations Across Malignancies Varying by Immunogenic Potential and the Likelihood of Response to Immunotherapy

by Islam Eljilany, Sam Coleman, Aik Choon Tan, Martin D. McCarter, John Carpten, Howard Colman, Abdul Rafeh Naqash, Igor Puzanov, Susanne M. Arnold, Michelle L. Churchman, Daniel Spakowicz, Bodour Salhia, Julian A. Marin-Acevedo, Shridar Ganesan, Aakrosh Ratan, Craig Shriver, Patrick Hwu, William S. Dalton, George J. Weiner, Jose R. Conejo-Garcia, Paulo Rodriguez and Ahmad A. Tarhini Show full author list Hide full author list

Cells 2024, 13(23), 1993; https://doi.org/10.3390/cells13231993 - 3 Dec 2024

Cited by 1 | Viewed by 2227

Abstract

Background: Solid tumors vary by the immunogenic potential of the tumor microenvironment (TME) and the likelihood of response to immunotherapy. The emerging literature has identified key immune cell populations that significantly impact immune activation or suppression within the TME. This study investigated candidate [...] Read more.

Background: Solid tumors vary by the immunogenic potential of the tumor microenvironment (TME) and the likelihood of response to immunotherapy. The emerging literature has identified key immune cell populations that significantly impact immune activation or suppression within the TME. This study investigated candidate T-cell populations and their differential infiltration within different tumor types as estimated from mRNA co-expression levels of the corresponding cellular markers. Methods: We analyzed the mRNA co-expression levels of cellular biomarkers that define stem-like tumor-infiltrating lymphocytes (TILs), tissue-resident memory T-cells (TRM), early dysfunctional T-cells, late dysfunctional T-cells, activated-potentially anti-tumor (APA) T-cells and Butyrophilin 3A (BTN3A) isoforms, utilizing clinical and transcriptomic data from 1892 patients diagnosed with melanoma, bladder, ovarian, or pancreatic carcinomas. Real-world data were collected under the Total Cancer Care Protocol and the Avatar^® project (NCT03977402) across 18 cancer centers. Furthermore, we compared the survival outcomes following immune checkpoint inhibitors (ICIs) based on immune cell gene expression. Results: In melanoma and bladder cancer, the estimated infiltration of APA T-cells differed significantly (p = 4.67 × 10⁻¹² and p = 5.80 × 10⁻¹², respectively) compared to ovarian and pancreatic cancers. Ovarian cancer had lower TRM T-cell infiltration than melanoma, bladder, and pancreatic (p = 2.23 × 10⁻⁸, 3.86 × 10⁻²⁸, and 7.85 × 10⁻⁹, respectively). Similar trends were noted with stem-like, early, and late dysfunctional T-cells. Melanoma and ovarian expressed BTN3A isoforms more than other malignancies. Higher densities of stem-like TILs; TRM, early and late dysfunctional T-cells; APA T-cells; and BTN3A isoforms were associated with increased survival in melanoma (p = 0.0075, 0.00059, 0.013, 0.005, 0.0016, and 0.041, respectively). The TRM gene signature was a moderate predictor of survival in the melanoma cohort (AUROC = 0.65), with similar findings in testing independent public datasets of ICI-treated patients with melanoma (AUROC 0.61–0.64). Conclusions: Key cellular elements related to immune activation are more heavily infiltrated within ICI-responsive versus non-responsive malignancies, supporting a central role in anti-tumor immunity. In melanoma patients treated with ICIs, higher densities of stem-like TILs, TRM T-cells, early dysfunctional T-cells, late dysfunctional T-cells, APA T-cells, and BTN3A isoforms were associated with improved survival. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms in Immune Regulation)

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18 pages, 2732 KiB

Open AccessEditor’s ChoiceArticle

Deficiencies of Inducible Costimulator (ICOS) During Chronic Infection with Toxoplasma gondii Upregulate the CD28-Dependent Cytotoxicity of CD8⁺ T Cells and Their Effector Function Against Tissue Cysts of the Parasite

by Rajesh Mani, Kanal E. Balu and Yasuhiro Suzuki

Cells 2024, 13(23), 1998; https://doi.org/10.3390/cells13231998 - 3 Dec 2024

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Abstract

We recently identified that the cerebral mRNA expression of inducible costimulator (ICOS) and its ligand, ICOSL, both significantly increase during the elimination of Toxoplasma gondii cysts from the brains of infected mice by the perforin-mediated cytotoxic activity of CD8⁺ T cells. In [...] Read more.

We recently identified that the cerebral mRNA expression of inducible costimulator (ICOS) and its ligand, ICOSL, both significantly increase during the elimination of Toxoplasma gondii cysts from the brains of infected mice by the perforin-mediated cytotoxic activity of CD8⁺ T cells. In the present study, we examined the role of ICOS in activating the effector activity of CD8⁺ T cells in response to the presence of cysts in infected mice. Following the adoptive transfer of splenic CD8⁺ T cells from chronically infected ICOS-deficient (ICOS^−/−) and wild-type (WT) mice to infected SCID mice, fewer CD8⁺ T cells were detected in the brains of the recipients of ICOS^−/− CD8⁺ T cells than the recipients of WT CD8⁺ T cells. Interestingly, even with the lower migration rate of the ICOS^−/− CD8⁺ T cells, those T cells eliminated T. gondii cysts more efficiently than WT CD8⁺ T cells did in the brains of the recipient mice. Consistently, the ICOS^−/− CD8⁺ T cells secreted greater amounts of granzyme B in response to T. gondii antigens in vitro than WT CD8⁺ T cells did. We identified that CD8⁺ T cells of infected ICOS^−/− mice express significantly greater levels of CD28 on their surface than CD8⁺ T cells of infected WT mice, and the relative expression of CD28 mRNA to CD8β mRNA levels in the brains of the recipients of those CD8⁺ T cells were strongly correlated with their relative expression levels of mRNA for T-bet transcription factors and perforin. Furthermore, blocking CD28 signaling using a combination of anti-CD80 and anti-CD86 antibodies eliminated the increased cytotoxic activity of the ICOS^−/− CD8⁺ T cells in vitro. The present study uncovered notable compensatory interactions between ICOS and CD28, which protected the cytotoxic effector activity of CD8⁺ T cells against microbial infection in a murine model of chronic infection with T. gondii. Full article

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20 pages, 18147 KiB

Open AccessEditor’s ChoiceArticle

CD38 Inhibitor 78c Attenuates Pro-Inflammatory Cytokine Expression and Osteoclastogenesis in Macrophages

by William Lory, Nityananda Chowdhury, Bridgette Wellslager, Subramanya Pandruvada, Yan Huang, Özlem Yilmaz and Hong Yu

Cells 2024, 13(23), 1971; https://doi.org/10.3390/cells13231971 - 28 Nov 2024

Cited by 2 | Viewed by 1737

Abstract

CD38, a nicotinamide adenine dinucleotide (NAD⁺) glycohydrolase, increases during infection or inflammation. Therefore, we aimed to evaluate the effects of a CD38 inhibitor (78c) on NAD⁺ levels, IL-1β, IL-6, TNF-α cytokine expressions, and osteoclastogenesis. The results show that treatment with [...] Read more.

CD38, a nicotinamide adenine dinucleotide (NAD⁺) glycohydrolase, increases during infection or inflammation. Therefore, we aimed to evaluate the effects of a CD38 inhibitor (78c) on NAD⁺ levels, IL-1β, IL-6, TNF-α cytokine expressions, and osteoclastogenesis. The results show that treatment with 78c on murine BMMs dose-dependently reduced CD38, reversed the decline of NAD⁺, and inhibited IL-1β, IL-6, and TNF-α pro-inflammatory cytokine levels induced by oral pathogen Porphyromonas gingivalis (Pg) or Aggregatibacter actinomycetemcomitans (Aa) or by advanced glycation end products (AGEs). Additionally, treatment with 78c dose-dependently suppressed osteoclastogenesis and bone resorption induced by RANKL. Treatment with 78c suppressed CD38, nuclear factor kappa-B (NF-κB), phosphoinositide 3-kinase (PI3K), and mitogen-activated protein kinases (MAPKs) induced by Pg, Aa, or AGEs, and suppressed podosome components (PI3K, Pyk2, Src, F-actin, integrins, paxillin, and talin) induced by RANKL. These results from our studies support the finding that the inhibition of CD38 by 78c is a promising therapeutic strategy to treat inflammatory bone loss diseases. However, treatment with a CD38 shRNA only significantly reduced IL-1β, IL-6, and TNF-α pro-inflammatory cytokine levels induced by AGEs. Compared with controls, it had limited effects on cytokine levels induced by Pg or Aa. Treatment with the CD38 shRNA enhanced RANKL-induced osteoclastogenesis, suggesting that 78c has some off-target effects. Full article

(This article belongs to the Collection Cell Biology in the United States: Latest Advances and Perspectives)

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30 pages, 7913 KiB

Open AccessEditor’s ChoiceArticle

Peripheral Blood Leukocyte Subpopulation Changes in Reaction to an Acute Psychosocial Stressor as Compared to an Active Placebo-Stressor in Healthy Young Males: Mediating Effects of Major Stress-Reactive Endocrine Parameters

by Lisa-Marie Walther, Angelina Gideon, Christine Sauter, Marcel Leist and Petra H. Wirtz

Cells 2024, 13(23), 1941; https://doi.org/10.3390/cells13231941 - 22 Nov 2024

Viewed by 1216

Abstract

Psychosocial stress has been proposed to induce a redistribution of immune cells, but a comparison with an active placebo-psychosocial stress control condition is lacking so far. We investigated immune cell redistribution due to psychosocial stress compared to that resulting from an active placebo-psychosocial [...] Read more.

Psychosocial stress has been proposed to induce a redistribution of immune cells, but a comparison with an active placebo-psychosocial stress control condition is lacking so far. We investigated immune cell redistribution due to psychosocial stress compared to that resulting from an active placebo-psychosocial stress but otherwise identical control condition. Moreover, we tested for mediating effects of endocrine parameters and blood volume changes. The final study sample comprised 64 healthy young men who underwent either a psychosocial stress condition (Trier Social Stress Test; TSST; n = 38) or an active placebo-psychosocial stress control condition (PlacTSST; n = 26). Immune cell counts and hemoglobin, epinephrine, norepinephrine, ACTH, renin, and aldosterone levels, as well as those of saliva cortisol, were determined before and up to 30 min after the TSST/PlacTSST. The TSST induced greater increases in total leukocyte, monocyte, and lymphocyte levels as compared to the PlacTSST (p’s ≤ 0.001), but in not granulocyte counts. Neutrophil granulocyte counts increased in reaction to both the TSST and PlacTSST (p’s ≤ 0.001), while eosinophil and basophil granulocyte counts did not. The psychosocial stress-induced increases in immune cell counts from baseline to peak (i.e., +1 min after TSST cessation) were independently mediated by parallel increases in epinephrine (ab’s ≤ −0.43; 95% CIs [LLs ≤ −0.66; ULs ≤ −0.09]). Subsequent decreases in immune cell counts from +1 min to +10 min after psychosocial stress cessation were mediated by parallel epinephrine, renin, and blood volume decreases (ab’s ≥ 0.17; 95% CIs [LLs ≥ 0.02; ULs ≥ 0.35]). Our findings indicate that psychosocial stress specifically induces immune cell count increases in most leukocyte subpopulations that are not secondary to the physical or cognitive demands of the stress task. Increases in the number of circulating neutrophil granulocytes, however, are not psychosocial stress-specific and even occur in situations with a low probability of threat or harm. Our findings point to a major role of epinephrine in mediating stress-induced immune cell count increases and of epinephrine, renin, and blood volume changes in mediating subsequent immune cell count decreases from +1 min to +10 min after psychosocial stress cessation. Full article

(This article belongs to the Special Issue Innate Immunity in Health and Disease)

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22 pages, 4829 KiB

Open AccessEditor’s ChoiceArticle

Host Genetics Background Affects Intestinal Cancer Development Associated with High-Fat Diet-Induced Obesity and Type 2 Diabetes

by Aya Ghnaim, Kareem Midlej, Osayd Zohud, Sama Karram, Arne Schaefer, Yael Houri-Haddad, Iqbal M. Lone and Fuad A. Iraqi

Cells 2024, 13(21), 1805; https://doi.org/10.3390/cells13211805 - 31 Oct 2024

Cited by 2 | Viewed by 1195

Abstract

Background: Obesity and type 2 diabetes (T2D) promote inflammation, increasing the risk of colorectal cancer (CRC). High-fat diet (HFD)-induced obesity is key to these diseases through biological mechanisms. This study examined the impact of genetic background on the multimorbidity of intestinal cancer, T2D, [...] Read more.

Background: Obesity and type 2 diabetes (T2D) promote inflammation, increasing the risk of colorectal cancer (CRC). High-fat diet (HFD)-induced obesity is key to these diseases through biological mechanisms. This study examined the impact of genetic background on the multimorbidity of intestinal cancer, T2D, and inflammation due to HFD-induced obesity. Methods: A cohort of 357 Collaborative Cross (CC) mice from 15 lines was fed either a control chow diet (CHD) or HFD for 12 weeks. Body weight was tracked biweekly, and blood glucose was assessed at weeks 6 and 12 via intraperitoneal glucose tolerance tests (IPGTT). At the study’s endpoint, intestinal polyps were counted, and cytokine profiles were analyzed to evaluate the inflammatory response. Results: HFD significantly increased blood glucose levels and body weight, with males showing higher susceptibility to T2D and obesity. Genetic variation across CC lines influenced glucose metabolism, body weight, and polyp development. Mice on HFD developed more intestinal polyps, with males showing higher counts than females. Cytokine analysis revealed diet-induced variations in pro-inflammatory markers like IL-6, IL-17A, and TNF-α, differing by genetic background and sex. Conclusions: Host genetics plays a crucial role in susceptibility to HFD-induced obesity, T2D, CRC, and inflammation. Genetic differences across CC lines contributed to variability in disease outcomes, providing insight into the genetic underpinnings of multimorbidity. This study supports gene-mapping efforts to develop personalized prevention and treatment strategies for these diseases. Full article

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28 pages, 7251 KiB

Open AccessEditor’s ChoiceArticle

The Power of Reagent Titration in Flow Cytometry

by Diana L. Bonilla, Alberta Paul, Jesus Gil-Pulido, Lily M. Park and Maria C. Jaimes

Cells 2024, 13(20), 1677; https://doi.org/10.3390/cells13201677 - 11 Oct 2024

Cited by 3 | Viewed by 8493

Abstract

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and [...] Read more.

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration–response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

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17 pages, 2965 KiB

Open AccessEditor’s ChoiceArticle

Systematic Comparison of CRISPR and shRNA Screens to Identify Essential Genes Using a Graph-Based Unsupervised Learning Model

by Yulian Ding, Connor Denomy, Andrew Freywald, Yi Pan, Franco J. Vizeacoumar, Frederick S. Vizeacoumar and Fang-Xiang Wu

Cells 2024, 13(19), 1653; https://doi.org/10.3390/cells13191653 - 4 Oct 2024

Cited by 1 | Viewed by 1844

Abstract

Generally, essential genes identified using shRNA and CRISPR are not always the same, raising questions about the choice between these two screening platforms. To address this, we systematically compared the performance of CRISPR and shRNA to identify essential genes across different gene expression [...] Read more.

Generally, essential genes identified using shRNA and CRISPR are not always the same, raising questions about the choice between these two screening platforms. To address this, we systematically compared the performance of CRISPR and shRNA to identify essential genes across different gene expression levels in 254 cell lines. As both platforms have a notable false positive rate, to correct this confounding factor, we first developed a graph-based unsupervised machine learning model to predict common essential genes. Furthermore, to maintain the unique characteristics of individual cell lines, we intersect essential genes derived from the biological experiment with the predicted common essential genes. Finally, we employed statistical methods to compare the ability of these two screening platforms to identify essential genes that exhibit differential expression across various cell lines. Our analysis yielded several noteworthy findings: (1) shRNA outperforms CRISPR in the identification of lowly expressed essential genes; (2) both screening methodologies demonstrate strong performance in identifying highly expressed essential genes but with limited overlap, so we suggest using a combination of these two platforms for highly expressed essential genes; (3) notably, we did not observe a single gene that becomes universally essential across all cancer cell lines. Full article

(This article belongs to the Collection Exclusive Papers from Editorial Board Members and Invited Scholars in Cells)

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16 pages, 4211 KiB

Open AccessEditor’s ChoiceArticle

An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines

by Akeem Sanni, Andrew I. Bennett, Yifan Huang, Isabella Gidi, Moyinoluwa Adeniyi, Judith Nwaiwu, Min H. Kang, Michelle E. Keyel, ChongFeng Gao, C. Patrick Reynolds, Brian Haab and Yehia Mechref

Cells 2024, 13(19), 1640; https://doi.org/10.3390/cells13191640 - 2 Oct 2024

Viewed by 3148

Abstract

Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell–cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic [...] Read more.

Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell–cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography–mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R² = 0.9961–0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types. Full article

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11 pages, 1339 KiB

Open AccessEditor’s ChoiceCommunication

Screening a Compound Library to Identify Additives That Boost Cytochrome P450 Enzyme Function in Vascularised Liver Spheres

by Baltasar Lucendo-Villarin, Yu Wang, Sunil K. Mallanna, Erin A. Kimbrel and David C. Hay

Cells 2024, 13(18), 1594; https://doi.org/10.3390/cells13181594 - 22 Sep 2024

Cited by 1 | Viewed by 1686

Abstract

To accurately study human organ function and disease ‘in the dish’, it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver, which is the largest solid organ in the body. The liver is a [...] Read more.

To accurately study human organ function and disease ‘in the dish’, it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver, which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however, severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore, the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study, we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore, we screened a compound library to identify additives that improved the maturity of in vitro-engineered human tissue, with the rationale that by taking such an approach, we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue. Full article

(This article belongs to the Collection Interdisciplinary Approaches to Studying Human Liver Biology and Promoting Organ Regeneration: Understanding the Curse of Prometheus)

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24 pages, 31715 KiB

Open AccessEditor’s ChoiceArticle

A Flow Cytometry-Based Examination of the Mouse White Blood Cell Differential in the Context of Age and Sex

by Elise Arlt, Andrea Kindermann, Anne-Kristin Fritsche, Alexander Navarrete Santos, Heike Kielstein and Ivonne Bazwinsky-Wutschke

Cells 2024, 13(18), 1583; https://doi.org/10.3390/cells13181583 - 20 Sep 2024

Cited by 3 | Viewed by 6129

Abstract

Analysis of the white blood cell differential as part of a flow cytometry-based approach is a common routine diagnostic tool used in clinics and research. For human blood, the methodological approach, suitable markers, and gating strategies are well-established. However, there is a lack [...] Read more.

Analysis of the white blood cell differential as part of a flow cytometry-based approach is a common routine diagnostic tool used in clinics and research. For human blood, the methodological approach, suitable markers, and gating strategies are well-established. However, there is a lack of information regarding the mouse blood count. In this article, we deliver a fast and easy protocol for reprocessing mouse blood for the purpose of flow cytometric analysis, as well as suitable markers and gating strategies. We also present two possible applications: for the analysis of the whole blood count, with blood from a cardiac puncture, and for the analysis of a certain leukocyte subset at multiple time points in the framework of a mouse experiment, using blood from the facial vein. Additionally, we provide orientation values by applying the method to 3-month-old and 24-month-old male and female C57BL/6J mice. Our analyses demonstrate differences in the leukocyte fractions depending on age and sex. We discuss the influencing factors and limitations that can affect the results and that, therefore, need to be considered when applying this method. The present study fills the gap in the knowledge related to the rare information on flow cytometric analysis of mouse blood and, thus, lays the foundation for further investigations in this area. Full article

(This article belongs to the Special Issue Flow Cytometry in Immunology Research)

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15 pages, 2800 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Importance of Transcript Variants in Transcriptome Analyses

by Kevin Vo, Yashica Sharma, Anohita Paul, Ryan Mohamadi, Amelia Mohamadi, Patrick E. Fields and M. A. Karim Rumi

Cells 2024, 13(17), 1502; https://doi.org/10.3390/cells13171502 - 8 Sep 2024

Cited by 4 | Viewed by 2214

Abstract

RNA sequencing (RNA-Seq) has become a widely adopted technique for studying gene expression. However, conventional RNA-Seq analyses rely on gene expression (GE) values that aggregate all the transcripts produced under a single gene identifier, overlooking the complexity of transcript variants arising from different [...] Read more.

RNA sequencing (RNA-Seq) has become a widely adopted technique for studying gene expression. However, conventional RNA-Seq analyses rely on gene expression (GE) values that aggregate all the transcripts produced under a single gene identifier, overlooking the complexity of transcript variants arising from different transcription start sites or alternative splicing. Transcript variants may encode proteins with diverse functional domains, or noncoding RNAs. This study explored the implications of neglecting transcript variants in RNA-Seq analyses. Among the 1334 transcription factor (TF) genes expressed in mouse embryonic stem (ES) or trophoblast stem (TS) cells, 652 were differentially expressed in TS cells based on GE values (365 upregulated and 287 downregulated, ≥absolute 2-fold changes, false discovery rate (FDR) p-value ≤ 0.05). The 365 upregulated genes expressed 883 transcript variants. Further transcript expression (TE) based analyses identified only 174 (<20%) of the 883 transcripts to be upregulated. The remaining 709 transcripts were either downregulated or showed no significant changes. Meanwhile, the 287 downregulated genes expressed 856 transcript variants and only 153 (<20%) of the 856 transcripts were downregulated. The other 703 transcripts were either upregulated or showed no significant change. Additionally, the 682 insignificant TF genes (GE values < absolute 2-fold changes and/or FDR p-values > 0.05) between ES and TS cells expressed 2215 transcript variants. These included 477 (>21%) differentially expressed transcripts (276 upregulated and 201 downregulated, ≥absolute 2-fold changes, FDR p-value ≤ 0.05). Hence, GE based RNA-Seq analyses do not represent accurate expression levels due to divergent transcripts expression from the same gene. Our findings show that by including transcript variants in RNA-Seq analyses, we can generate a precise understanding of a gene’s functional and regulatory landscape; ignoring the variants may result in an erroneous interpretation. Full article

(This article belongs to the Section Cell and Gene Therapy)

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24 pages, 8061 KiB

Open AccessEditor’s ChoiceArticle

TRIM44, a Novel Prognostic Marker, Supports the Survival of Proteasome-Resistant Multiple Myeloma Cells

by Trung Vu, Yuqin Wang, Annaliese Fowler, Anton Simieou and Nami McCarty

Cells 2024, 13(17), 1431; https://doi.org/10.3390/cells13171431 - 26 Aug 2024

Cited by 5 | Viewed by 1972

Abstract

TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a [...] Read more.

TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a Multiple Myeloma Research Foundation (MMRF) cohort of 858 patients, persisting across primary and recurrent MM cases. TRIM44 expression notably increases in advanced MM stages, indicating its potential role in disease progression. Single-cell RNA sequencing across MM stages showed significant TRIM44 upregulation in smoldering MM (SMM) and MM compared to normal bone marrow, especially in patients with t(4;14) cytogenetic abnormalities. This analysis further identified high TRIM44 expression as predictive of lower responsiveness to proteasome inhibitor (PI) treatments, underscoring its critical function in the unfolded protein response (UPR) in TRIM44-high MM cells. Our findings also demonstrate that TRIM44 facilitates SQSTM1 oligomerization under oxidative stress, essential for its phosphorylation and subsequent autophagic degradation. This process supports the survival of PI-resistant MM cells by activating the NRF2 pathway, which is crucial for oxidative stress response and, potentially, other chemotherapy-induced stressors. Additionally, TRIM44 counters the TRIM21-mediated suppression of the antioxidant response, enhancing MM cell survival under oxidative stress. Collectively, our discoveries highlight TRIM44’s significant role in MM progression and resistance to therapy, suggesting its potential value as a therapeutic target. Full article

(This article belongs to the Section Autophagy)

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20 pages, 4969 KiB

Open AccessEditor’s ChoiceArticle

The Expression of Toll-like Receptors in Cartilage Endplate Cells: A Role of Toll-like Receptor 2 in Pro-Inflammatory and Pro-Catabolic Gene Expression

by Tamara Mengis, Laura Bernhard, Andrea Nüesch, Irina Heggli, Nick Herger, Jan Devan, Roy Marcus, Christoph J. Laux, Florian Brunner, Mazda Farshad, Oliver Distler, Christine L. Le Maitre and Stefan Dudli

Cells 2024, 13(17), 1402; https://doi.org/10.3390/cells13171402 - 23 Aug 2024

Cited by 3 | Viewed by 1749

Abstract

Introduction: The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated [...] Read more.

Introduction: The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression. Methods: Gene expression of TLR1–10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry. Results: Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model. Conclusions: The expression of TLR1–10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC. Full article

(This article belongs to the Special Issue Cellular and Molecular Events in Intervertebral Disc Development, Homeostasis and Disease)

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14 pages, 5113 KiB

Open AccessEditor’s ChoiceArticle

Extracellular Vesicle Transplantation Is Beneficial for Acute Kidney Injury

by Amankeldi A. Salybekov, Shigeaki Okamura, Takayasu Ohtake, Sumi Hidaka, Takayuki Asahara and Shuzo Kobayashi

Cells 2024, 13(16), 1335; https://doi.org/10.3390/cells13161335 - 12 Aug 2024

Cited by 1 | Viewed by 1679

Abstract

Under vasculogenic conditioning, certain pro-inflammatory subsets within peripheral blood mononuclear cells (PBMCs) undergo phenotypic transformation into pro-regenerative types, such as vasculogenic endothelial progenitor cells, M2 macrophages, and regulatory T cells. These transformed cells are collectively termed regeneration-associated cells (RACs). In this study, we [...] Read more.

Under vasculogenic conditioning, certain pro-inflammatory subsets within peripheral blood mononuclear cells (PBMCs) undergo phenotypic transformation into pro-regenerative types, such as vasculogenic endothelial progenitor cells, M2 macrophages, and regulatory T cells. These transformed cells are collectively termed regeneration-associated cells (RACs). In this study, we aimed to investigate the therapeutic efficacy of RAC-derived extracellular vesicles (RACev) compared with a vehicle-treated group in the context of renal ischemia-reperfusion injury (R-IRI). Human PBMCs were cultured with defined growth factor cocktails for seven days to harvest RACs. EV quantity and size were characterized by nanoparticle tracking analysis. Notably, the systemic injection of RACev significantly decreased serum creatinine and blood urine nitrogen at day three compared to the control group. Histologically, the treatment group showed less fibrosis in the cortex and medullary areas (p < 0.04 and p < 0.01) compared to the control group. The CD31 staining confirmed enhanced capillary densities in the treatment group compared to the control group (p < 0.003). These beneficial effects were accompanied by angiogenesis, anti-fibrosis, anti-inflammation, and anti-apoptosis RACev miR delivery to ischemic injury to control inflammatory, endothelial mesenchymal transition, and hypoxia pathways. In vivo bioluminescence analysis demonstrated a preferential accumulation of RACev in the IR-injured kidney. The systemic transplantation of RACev beneficially restored kidney function by protecting from tissue fibrosis and through anti-inflammation, angiogenesis, and anti-apoptosis miR delivery to the ischemic tissue. Full article

(This article belongs to the Special Issue Extracellular Vesicles in Tissue Repair and Regeneration)

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12 pages, 1822 KiB

Open AccessEditor’s ChoiceArticle

Synthetic mRNAs Containing Minimalistic Untranslated Regions Are Highly Functional In Vitro and In Vivo

by Shahab Mamaghani, Rocco Roberto Penna, Julia Frei, Conrad Wyss, Mark Mellett, Thomas Look, Tobias Weiss, Emmanuella Guenova, Thomas M. Kündig, Severin Lauchli and Steve Pascolo

Cells 2024, 13(15), 1242; https://doi.org/10.3390/cells13151242 - 24 Jul 2024

Cited by 6 | Viewed by 4227

Abstract

Synthetic mRNA produced by in vitro transcription (ivt mRNA) is the active pharmaceutical ingredient of approved anti-COVID-19 vaccines and of many drugs under development. Such synthetic mRNA typically contains several hundred bases of non-coding “untranslated” regions (UTRs) that are involved in the stabilization [...] Read more.

Synthetic mRNA produced by in vitro transcription (ivt mRNA) is the active pharmaceutical ingredient of approved anti-COVID-19 vaccines and of many drugs under development. Such synthetic mRNA typically contains several hundred bases of non-coding “untranslated” regions (UTRs) that are involved in the stabilization and translation of the mRNA. However, UTRs are often complex structures, which may complicate the entire production process. To eliminate this obstacle, we managed to reduce the total amount of nucleotides in the UTRs to only four bases. In this way, we generate minimal ivt mRNA (“minRNA”), which is less complex than the usual optimized ivt mRNAs that are contained, for example, in approved vaccines. We have compared the efficacy of minRNA to common augmented mRNAs (with UTRs of globin genes or those included in licensed vaccines) in vivo and in vitro and could demonstrate equivalent functionalities. Our minimal mRNA design will facilitate the further development and implementation of ivt mRNA-based vaccines and therapies. Full article

(This article belongs to the Section Cell and Gene Therapy)

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17 pages, 3144 KiB

Open AccessEditor’s ChoiceArticle

Advanced Maternal Age Differentially Affects Embryonic Tissues with the Most Severe Impact on the Developing Brain

by Caroline Kokorudz, Bethany N. Radford, Wendy Dean and Myriam Hemberger

Cells 2023, 12(1), 76; https://doi.org/10.3390/cells12010076 - 24 Dec 2022

Cited by 7 | Viewed by 3423

Abstract

Advanced maternal age (AMA) poses the single greatest risk to a successful pregnancy. Apart from the impact of AMA on oocyte fitness, aged female mice often display defects in normal placentation. Placental defects in turn are tightly correlated with brain and cardiovascular abnormalities. [...] Read more.

Advanced maternal age (AMA) poses the single greatest risk to a successful pregnancy. Apart from the impact of AMA on oocyte fitness, aged female mice often display defects in normal placentation. Placental defects in turn are tightly correlated with brain and cardiovascular abnormalities. It therefore follows that placenta, brain and heart development may be particularly susceptible to the impact of AMA. In the current study, we compared global transcriptomes of placentas, brains, hearts, and facial prominences from mid-gestation mouse conceptuses developed in young control (7–13 wks) and aging (43–50 wks) females. We find that AMA increases transcriptional heterogeneity in all tissues, but particularly in fetal brain. Importantly, even overtly normally developed embryos from older females display dramatic expression changes in neurodevelopmental genes. These transcriptomic alterations in the brain are likely induced by defects in placental development. Using trophoblast stem cells (TSCs) as a model, we show that exposure to aging uterine stromal cell-conditioned medium interferes with normal TSC proliferation and causes precocious differentiation, recapitulating many of the defects observed in placentas from aged females. These data highlight the increased risk of AMA on reproductive outcome, with neurodevelopment being the most sensitive to such early perturbations and with potential for lifelong impact. Full article

(This article belongs to the Section Reproductive Cells and Development)

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20 pages, 3401 KiB

Open AccessEditor’s ChoiceArticle

Impact of Mitochondrial A3243G Heteroplasmy on Mitochondrial Bioenergetics and Dynamics of Directly Reprogrammed MELAS Neurons

by Dar-Shong Lin, Yu-Wen Huang, Che-Sheng Ho, Tung-Sun Huang, Tsung-Han Lee, Tsu-Yen Wu, Zon-Darr Huang and Tuan-Jen Wang

Cells 2023, 12(1), 15; https://doi.org/10.3390/cells12010015 - 21 Dec 2022

Cited by 10 | Viewed by 3079

Abstract

The MELAS syndrome primarily affecting the CNS is mainly caused by the m.A3243G mutation. The heteroplasmy in different tissues affects the phenotypic spectrum, yet the impact of various levels of m.A3243G heteroplasmy on CNS remains elusive due to the lack of a proper [...] Read more.

The MELAS syndrome primarily affecting the CNS is mainly caused by the m.A3243G mutation. The heteroplasmy in different tissues affects the phenotypic spectrum, yet the impact of various levels of m.A3243G heteroplasmy on CNS remains elusive due to the lack of a proper neuronal model harboring m.A3243G mutation. We generated induced neurons (iNs) through the direct reprogramming of MELAS patients, with derived fibroblasts harboring high (>95%), intermediate (68%), and low (20%) m.A3243G mutation. iNs demonstrated neuronal morphology with neurite outgrowth, branching, and dendritic spines. The heteroplasmy and deficiency of respiratory chain complexes were retained in MELAS iNs. High heteroplasmy elicited the elevation in ROS levels and the disruption of mitochondrial membrane potential. Furthermore, high and intermediate heteroplasmy led to the impairment of mitochondrial bioenergetics and a change in mitochondrial dynamics toward the fission and fragmentation of mitochondria, with a reduction in mitochondrial networks. Moreover, iNs derived from aged individuals manifested with mitochondrial fission. These results help us in understanding the impact of various heteroplasmic levels on mitochondrial bioenergetics and mitochondrial dynamics in neurons as the underlying pathomechanism of neurological manifestations of MELAS syndrome. Furthermore, these findings provide targets for further pharmacological approaches of mitochondrial diseases and validate iNs as a reliable platform for studies in neuronal aspects of aging, neurodegenerative disorders, and mitochondrial diseases. Full article

(This article belongs to the Special Issue Advances in Mitochondrial Dynamics and Neurodegeneration)

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28 pages, 5418 KiB

Open AccessEditor’s ChoiceArticle

Human PSEN1 Mutant Glia Improve Spatial Learning and Memory in Aged Mice

by Henna Jäntti, Minna Oksanen, Pinja Kettunen, Stella Manta, Lionel Mouledous, Hennariikka Koivisto, Johanna Ruuth, Kalevi Trontti, Hiramani Dhungana, Meike Keuters, Isabelle Weert, Marja Koskuvi, Iiris Hovatta, Anni-Maija Linden, Claire Rampon, Tarja Malm, Heikki Tanila, Jari Koistinaho and Taisia Rolova

Cells 2022, 11(24), 4116; https://doi.org/10.3390/cells11244116 - 18 Dec 2022

Cited by 3 | Viewed by 4169

Abstract

The PSEN1 ΔE9 mutation causes a familial form of Alzheimer’s disease (AD) by shifting the processing of amyloid precursor protein (APP) towards the generation of highly amyloidogenic Aβ42 peptide. We have previously shown that the PSEN1 ΔE9 mutation in human-induced pluripotent stem cell [...] Read more.

The PSEN1 ΔE9 mutation causes a familial form of Alzheimer’s disease (AD) by shifting the processing of amyloid precursor protein (APP) towards the generation of highly amyloidogenic Aβ42 peptide. We have previously shown that the PSEN1 ΔE9 mutation in human-induced pluripotent stem cell (iPSC)-derived astrocytes increases Aβ42 production and impairs cellular responses. Here, we injected PSEN1 ΔE9 mutant astrosphere-derived glial progenitors into newborn mice and investigated mouse behavior at the ages of 8, 12, and 16 months. While we did not find significant behavioral changes in younger mice, spatial learning and memory were paradoxically improved in 16-month-old PSEN1 ΔE9 glia-transplanted male mice as compared to age-matched isogenic control-transplanted animals. Memory improvement was associated with lower levels of soluble, but not insoluble, human Aβ42 in the mouse brain. We also found a decreased engraftment of PSEN1 ΔE9 mutant cells in the cingulate cortex and significant transcriptional changes in both human and mouse genes in the hippocampus, including the extracellular matrix-related genes. Overall, the presence of PSEN1 ΔE9 mutant glia exerted a more beneficial effect on aged mouse brain than the isogenic control human cells likely as a combination of several factors. Full article

(This article belongs to the Special Issue Glial Cells in Synaptic Plasticity)

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13 pages, 2045 KiB

Open AccessEditor’s ChoiceArticle

Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages

by Riley J. Morrow, Amr H. Allam, Josh Konecnik, David Baloyan, Christine Dijkstra, Moritz F. Eissmann, Saumya P. Jacob, Megan O’Brien, Ashleigh R. Poh and Matthias Ernst

Cells 2022, 11(24), 4104; https://doi.org/10.3390/cells11244104 - 17 Dec 2022

Viewed by 2495

Abstract

Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, [...] Read more.

Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, including macrophages and other myeloid cells. Here, we benchmark Myc inactivation in tumor cells against the contribution of its expression in myeloid cells of murine hosts that harbor endogenous or allograft tumors. Surprisingly, we observe that LysM^Cre-mediated Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade. Likewise, we find no evidence for Myc ablation to revert or antagonize the polarization of alternatively activated immunosuppressive macrophages. Thus, we surmise that systemic targeting of Myc activity may confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than reinvigorating the anti-tumor activity of macrophages. Full article

(This article belongs to the Special Issue MYC Signaling in Cancer)

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15 pages, 1461 KiB

Open AccessEditor’s ChoiceArticle

Pre-Transplant Prediction of Acute Graft-versus-Host Disease Using the Gut Microbiome

by Ramtin Zargari Marandi, Mette Jørgensen, Emma Elizabeth Ilett, Jens Christian Nørgaard, Marc Noguera-Julian, Roger Paredes, Jens D. Lundgren, Henrik Sengeløv and Cameron Ross MacPherson

Cells 2022, 11(24), 4089; https://doi.org/10.3390/cells11244089 - 16 Dec 2022

Cited by 5 | Viewed by 4001

Abstract

Gut microbiota is thought to influence host responses to allogeneic hematopoietic stem cell transplantation (aHSCT). Recent evidence points to this post-transplant for acute graft-versus-host disease (aGvHD). We asked whether any such association might be found pre-transplant and conducted a metagenome-wide association study (MWAS) [...] Read more.

Gut microbiota is thought to influence host responses to allogeneic hematopoietic stem cell transplantation (aHSCT). Recent evidence points to this post-transplant for acute graft-versus-host disease (aGvHD). We asked whether any such association might be found pre-transplant and conducted a metagenome-wide association study (MWAS) to explore. Microbial abundance profiles were estimated using ensembles of Kaiju, Kraken2, and DeepMicrobes calls followed by dimensionality reduction. The area under the curve (AUC) was used to evaluate classification of the samples (aGvHD vs. none) using an elastic net to test the relevance of metagenomic data. Clinical data included the underlying disease (leukemia vs. other hematological malignancies), recipient age, and sex. Among 172 aHSCT patients of whom 42 developed aGVHD post transplantation, a total of 181 pre-transplant tool samples were analyzed. The top performing model predicting risk of aGVHD included a reduced species profile (AUC = 0.672). Beta diversity (37% in Jaccard’s Nestedness by mean fold change, p < 0.05) was lower in those developing aGvHD. Ten bacterial species including Prevotella and Eggerthella genera were consistently found to associate with aGvHD in indicator species analysis, as well as relief and impurity-based algorithms. The findings support the hypothesis on potential associations between gut microbiota and aGvHD based on a data-driven approach to MWAS. This highlights the need and relevance of routine stool collection for the discovery of novel biomarkers. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Microenvironment)

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21 pages, 27931 KiB

Open AccessEditor’s ChoiceArticle

Loss of Pex1 in Inner Ear Hair Cells Contributes to Cochlear Synaptopathy and Hearing Loss

by Stephanie A. Mauriac, Thibault Peineau, Aamir Zuberi, Cathleen Lutz and Gwénaëlle S. G. Géléoc

Cells 2022, 11(24), 3982; https://doi.org/10.3390/cells11243982 - 9 Dec 2022

Cited by 3 | Viewed by 3293

Abstract

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause [...] Read more.

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1. Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1^Creor VGlut3^Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function. Full article

(This article belongs to the Special Issue Exclusive Review Papers in Autophagy)

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17 pages, 3633 KiB

Open AccessEditor’s ChoiceArticle

High-Content RNAi Phenotypic Screening Unveils the Involvement of Human Ubiquitin-Related Enzymes in Late Cytokinesis

by Mikaël Boullé, Laurianne Davignon, Keïs Nabhane Saïd Halidi, Salomé Guez, Emilie Giraud, Marcel Hollenstein and Fabrice Agou

Cells 2022, 11(23), 3862; https://doi.org/10.3390/cells11233862 - 30 Nov 2022

Cited by 1 | Viewed by 2764

Abstract

CEP55 is a central regulator of late cytokinesis and is overexpressed in numerous cancers. Its post-translationally controlled recruitment to the midbody is crucial to the structural coordination of the abscission sequence. Our recent evidence that CEP55 contains two ubiquitin-binding domains was the first [...] Read more.

CEP55 is a central regulator of late cytokinesis and is overexpressed in numerous cancers. Its post-translationally controlled recruitment to the midbody is crucial to the structural coordination of the abscission sequence. Our recent evidence that CEP55 contains two ubiquitin-binding domains was the first structural and functional link between ubiquitin signaling and ESCRT-mediated severing of the intercellular bridge. So far, high-content screens focusing on cytokinesis have used multinucleation as the endpoint readout. Here, we report an automated image-based detection method of intercellular bridges, which we applied to further our understanding of late cytokinetic signaling by performing an RNAi screen of ubiquitin ligases and deubiquitinases. A secondary validation confirmed four candidate genes, i.e., LNX2, NEURL, UCHL1 and RNF157, whose downregulation variably affects interconnected phenotypes related to CEP55 and its UBDs, as follows: decreased recruitment of CEP55 to the midbody, increased number of midbody remnants per cell, and increased frequency of intercellular bridges or multinucleation events. This brings into question the Notch-dependent or independent contributions of LNX2 and NEURL proteins to late cytokinesis. Similarly, the role of UCHL1 in autophagy could link its function with the fate of midbody remnants. Beyond the biological interest, this high-content screening approach could also be used to isolate anticancer drugs that act by impairing cytokinesis and CEP55 functions. Full article

(This article belongs to the Special Issue Molecular Factors and Mechanisms Involved in Cytokinesis II)

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13 pages, 2027 KiB

Open AccessEditor’s ChoiceArticle

4-Methylumebelliferone Enhances Radiosensitizing Effects of Radioresistant Oral Squamous Cell Carcinoma Cells via Hyaluronan Synthase 3 Suppression

by Kazuki Hasegawa, Ryo Saga, Kentaro Ohuchi, Yoshikazu Kuwahara, Kazuo Tomita, Kazuhiko Okumura, Tomoaki Sato, Manabu Fukumoto, Eichi Tsuruga and Yoichiro Hosokawa

Cells 2022, 11(23), 3780; https://doi.org/10.3390/cells11233780 - 25 Nov 2022

Cited by 6 | Viewed by 2317

Abstract

Radioresistant (RR) cells are poor prognostic factors for tumor recurrence and metastasis after radiotherapy. The hyaluronan (HA) synthesis inhibitor, 4-methylumbelliferone (4-MU), shows anti-tumor and anti-metastatic effects through suppressing HA synthase (HAS) expression in various cancer cells. We previously reported that the administration of [...] Read more.

Radioresistant (RR) cells are poor prognostic factors for tumor recurrence and metastasis after radiotherapy. The hyaluronan (HA) synthesis inhibitor, 4-methylumbelliferone (4-MU), shows anti-tumor and anti-metastatic effects through suppressing HA synthase (HAS) expression in various cancer cells. We previously reported that the administration of 4-MU with X-ray irradiation enhanced radiosensitization. However, an effective sensitizer for radioresistant (RR) cells is yet to be established, and it is unknown whether 4-MU exerts radiosensitizing effects on RR cells. We investigated the radiosensitizing effects of 4-MU in RR cell models. This study revealed that 4-MU enhanced intracellular oxidative stress and suppressed the expression of cluster-of-differentiation (CD)-44 and cancer stem cell (CSC)-like phenotypes. Interestingly, eliminating extracellular HA using HA-degrading enzymes did not cause radiosensitization, whereas HAS3 knockdown using siRNA showed similar effects as 4-MU treatment. These results suggest that 4-MU treatment enhances radiosensitization of RR cells through enhancing oxidative stress and suppressing the CSC-like phenotype. Furthermore, the radiosensitizing mechanisms of 4-MU may involve HAS3 or intracellular HA synthesized by HAS3. Full article

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17 pages, 7647 KiB

Open AccessEditor’s ChoiceArticle

Dynamic Changes in the Extracellular Matrix in Primary, Metastatic, and Recurrent Ovarian Cancers

by Arkadiusz Gertych, Ann E. Walts, Keyi Cheng, Manyun Liu, Joshi John, Jenny Lester, Beth Y. Karlan and Sandra Orsulic

Cells 2022, 11(23), 3769; https://doi.org/10.3390/cells11233769 - 25 Nov 2022

Cited by 10 | Viewed by 3257

Abstract

Cancer-associated fibroblasts (CAFs) and their extracellular matrix are active participants in cancer progression. While it is known that functionally different subpopulations of CAFs co-exist in ovarian cancer, it is unclear whether certain CAF subsets are enriched during metastatic progression and/or chemotherapy. Using computational [...] Read more.

Cancer-associated fibroblasts (CAFs) and their extracellular matrix are active participants in cancer progression. While it is known that functionally different subpopulations of CAFs co-exist in ovarian cancer, it is unclear whether certain CAF subsets are enriched during metastatic progression and/or chemotherapy. Using computational image analyses of patient-matched primary high-grade serous ovarian carcinomas, synchronous pre-chemotherapy metastases, and metachronous post-chemotherapy metastases from 42 patients, we documented the dynamic spatiotemporal changes in the extracellular matrix, fibroblasts, epithelial cells, immune cells, and CAF subsets expressing different extracellular matrix components. Among the different CAF subsets, COL11A1⁺ CAFs were associated with linearized collagen fibers and exhibited the greatest enrichment in pre- and post-chemotherapy metastases compared to matched primary tumors. Although pre- and post-chemotherapy metastases were associated with increased CD8⁺ T cell infiltration, the infiltrate was not always evenly distributed between the stroma and cancer cells, leading to an increased frequency of the immune-excluded phenotype where the majority of CD8⁺ T cells are present in the tumor stroma but absent from the tumor parenchyma. Overall, most of the differences in the tumor microenvironment were observed between primary tumors and metastases, while fewer differences were observed between pre- and post-treatment metastases. These data suggest that the tumor microenvironment is largely determined by the primary vs. metastatic location of the tumor while chemotherapy does not have a significant impact on the host microenvironment. Full article

(This article belongs to the Special Issue Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy)

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15 pages, 7082 KiB

Open AccessEditor’s ChoiceArticle

Low-Dose rIL-15 Protects from Nephrotoxic Serum Nephritis via CD8⁺ T Cells

by Agnes A. Mooslechner, Max Schuller, Katharina Artinger, Alexander H. Kirsch, Corinna Schabhüttl, Philipp Eller, Alexander R. Rosenkranz and Kathrin Eller

Cells 2022, 11(22), 3656; https://doi.org/10.3390/cells11223656 - 18 Nov 2022

Cited by 3 | Viewed by 2937

Abstract

Rapid progressive glomerulonephritis (GN) often leads to end-stage kidney disease, driving the need for renal replacement therapy and posing a global health burden. Low-dose cytokine-based immunotherapies provide a new strategy to treat GN. IL-15 is a strong candidate for the therapy of immune-mediated [...] Read more.

Rapid progressive glomerulonephritis (GN) often leads to end-stage kidney disease, driving the need for renal replacement therapy and posing a global health burden. Low-dose cytokine-based immunotherapies provide a new strategy to treat GN. IL-15 is a strong candidate for the therapy of immune-mediated kidney disease since it has proven to be tubular-protective before. Therefore, we set out to test the potential of low-dose rIL-15 treatment in a mouse model of nephrotoxic serum nephritis (NTS), mimicking immune complex-driven GN in humans. A single low-dose treatment with rIL-15 ameliorated NTS, reflected by reduced albuminuria, less tissue scarring, fewer myeloid cells in the kidney, and improved tubular epithelial cell survival. In addition, CD8⁺ T cells, a primary target of IL-15, showed altered gene expression and function corresponding with less cytotoxicity mediated by rIL-15. With the use of transgenic knock-out mice, antibody depletion, and adoptive cell transfer studies, we here show that the beneficial effects of rIL-15 treatment in NTS depended on CD8⁺ T cells, suggesting a pivotal role for them in the underlying mechanism. Our findings add to existing evidence of the association of IL-15 with kidney health and imply a potential for low-dose rIL-15 immunotherapies in GN. Full article

(This article belongs to the Special Issue Immune Mechanisms in Glomerulonephritis)

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15 pages, 2879 KiB

Open AccessEditor’s ChoiceArticle

The HSF1-CPT1a Pathway Is Differentially Regulated in NAFLD Progression

by Wiebke Breternitz, Friedrich Sandkühler, Frauke Grohmann, Jochen Hampe, Mario Brosch, Alexander Herrmann, Clemens Schafmayer, Christian Meinhardt, Stefan Schreiber, Alexander Arlt and Claudia Geismann

Cells 2022, 11(21), 3504; https://doi.org/10.3390/cells11213504 - 4 Nov 2022

Cited by 5 | Viewed by 3236

Abstract

Obesity and obesity-associated diseases represent one of the key health challenges of our time. In this context, aberrant hepatic lipid accumulation is a central pathological aspect of non-alcoholic fatty liver disease (NAFLD). By comparing methylation signatures of liver biopsies before and after bariatric [...] Read more.

Obesity and obesity-associated diseases represent one of the key health challenges of our time. In this context, aberrant hepatic lipid accumulation is a central pathological aspect of non-alcoholic fatty liver disease (NAFLD). By comparing methylation signatures of liver biopsies before and after bariatric surgery, we recently demonstrated the strong enrichment of differentially methylated heat shock factor 1 (HSF1) binding sites (>400-fold) in the process of liver remodeling, indicating a crucial role of HSF1 in modulating central aspects of NAFLD pathogenesis. Using cellular models of NAFLD, we were able to show that HSF1 is activated during fat accumulation in hepatocytes, mimicking conditions in patients before bariatric surgery. This induction was abolished by starving the cells, mimicking the situation after bariatric surgery. Regarding this connection, carnitine palmitoyltransferase 1 isoform A (CTP1a), a central regulator of lipid beta-oxidation, was identified as a HSF1 target gene by promoter analysis and HSF1 knockdown experiments. Finally, pharmacological activation of HSF1 through celastrol reduced fat accumulation in the cells in a HSF1-dependent manner. In conclusion, we were able to confirm the relevance of HSF1 activity and described a functional HSF1-CPT1a pathway in NAFLD pathogenesis. Full article

(This article belongs to the Special Issue Metabolic Regulation: Cell Growth and Proliferation)

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27 pages, 7126 KiB

Open AccessEditor’s ChoiceArticle

Dendritic Cells or Macrophages? The Microenvironment of Human Clear Cell Renal Cell Carcinoma Imprints a Mosaic Myeloid Subtype Associated with Patient Survival

by Dorothee Brech, Anna S. Herbstritt, Sarah Diederich, Tobias Straub, Evangelos Kokolakis, Martin Irmler, Johannes Beckers, Florian A. Büttner, Elke Schaeffeler, Stefan Winter, Matthias Schwab, Peter J. Nelson and Elfriede Noessner

Cells 2022, 11(20), 3289; https://doi.org/10.3390/cells11203289 - 19 Oct 2022

Cited by 10 | Viewed by 4673

Abstract

Since their initial description by Elie Metchnikoff, phagocytes have sparked interest in a variety of biologic disciplines. These important cells perform central functions in tissue repair and immune activation as well as tolerance. Myeloid cells can be immunoinhibitory, particularly in the tumor microenvironment, [...] Read more.

Since their initial description by Elie Metchnikoff, phagocytes have sparked interest in a variety of biologic disciplines. These important cells perform central functions in tissue repair and immune activation as well as tolerance. Myeloid cells can be immunoinhibitory, particularly in the tumor microenvironment, where their presence is generally associated with poor patient prognosis. These cells are highly adaptable and plastic, and can be modulated to perform desired functions such as antitumor activity, if key programming molecules can be identified. Human clear cell renal cell carcinoma (ccRCC) is considered immunogenic; yet checkpoint blockades that target T cell dysfunction have shown limited clinical efficacy, suggesting additional layers of immunoinhibition. We previously described “enriched-in-renal cell carcinoma” (erc) DCs that were often found in tight contact with dysfunctional T cells. Using transcriptional profiling and flow cytometry, we describe here that ercDCs represent a mosaic cell type within the macrophage continuum co-expressing M1 and M2 markers. The polarization state reflects tissue-specific signals that are characteristic of RCC and renal tissue homeostasis. ErcDCs are tissue-resident with increasing prevalence related to tumor grade. Accordingly, a high ercDC score predicted poor patient survival. Within the profile, therapeutic targets (VSIG4, NRP1, GPNMB) were identified with promise to improve immunotherapy. Full article

(This article belongs to the Special Issue In Honor of Nobel Prize in Physiology or Medicine: Cell-Mediated Immunity)

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11 pages, 1632 KiB

Open AccessEditor’s ChoiceArticle

Characterization of the Aquaporin-9 Inhibitor RG100204 In Vitro and in db/db Mice

by Marilina Florio, Angelica Engfors, Patrizia Gena, Jessica Larsson, Alessandro Massaro, Stella Timpka, Martina Kvist Reimer, Per Kjellbom, Eric Beitz, Urban Johanson, Michael Rützler and Giuseppe Calamita

Cells 2022, 11(19), 3118; https://doi.org/10.3390/cells11193118 - 4 Oct 2022

Cited by 9 | Viewed by 2829

Abstract

Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas [...] Read more.

Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10⁻⁸ M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function. Full article

(This article belongs to the Section Cellular Metabolism)

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17 pages, 5246 KiB

Open AccessEditor’s ChoiceArticle

Proteomic and Phosphoproteomic Profiling Reveals the Oncogenic Role of Protein Kinase D Family Kinases in Cholangiocarcinoma

by Yun Lu, Xiangyu Li, Kai Zhao, Yuanxin Shi, Zhengdong Deng, Wei Yao and Jianming Wang

Cells 2022, 11(19), 3088; https://doi.org/10.3390/cells11193088 - 30 Sep 2022

Cited by 7 | Viewed by 3058

Abstract

Cholangiocarcinoma (CCA) is a lethal malignancy in the hepatobiliary system, with dysregulated protein expression and phosphorylation signaling. However, the protein and phosphorylation signatures of CCAs are little-known. Here, we performed the proteomic and phosphoproteomic profiling of tumors and normal adjacent tissues (NATs) from [...] Read more.

Cholangiocarcinoma (CCA) is a lethal malignancy in the hepatobiliary system, with dysregulated protein expression and phosphorylation signaling. However, the protein and phosphorylation signatures of CCAs are little-known. Here, we performed the proteomic and phosphoproteomic profiling of tumors and normal adjacent tissues (NATs) from patients with CCA and predicted eleven PKs high-potentially related to CCA with a comprehensive inference of the functional protein kinases (PKs) (CifPK) pipeline. Besides the two known CCA-associated PKs, we screened the remaining candidates and uncovered five PKs as novel regulators in CCA. Specifically, the protein kinase D (PKD) family members, including PRKD1, PRKD2, and PRKD3, were identified as critical regulators in CCA. Moreover, the pan-inhibitor of the PKD family, 1-naphthyl PP1 (1-NA-PP1), was validated as a potent agent for inhibiting the proliferation, migration, and invasion ability of CCA cells. This study reveals new PKs associated with CCA and suggests PRKD kinases as novel treatment targets for CCA. Full article

(This article belongs to the Special Issue New Insights into Proteomics and Post-translational Modification of Proteins)

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20 pages, 3466 KiB

Open AccessEditor’s ChoiceArticle

Effects of Cigarette Smoke on Adipose and Skeletal Muscle Tissue: In Vivo and In Vitro Studies

by Lei Wang, Lieke E. J. van Iersel, Charlotte E. Pelgrim, Jingyi Lu, Ingrid van Ark, Thea Leusink-Muis, Harry R. Gosker, Ramon C. J. Langen, Annemie M. W. J. Schols, Josep M. Argilés, Ardy van Helvoort, Aletta D. Kraneveld, Johan Garssen, Paul A. J. Henricks, Gert Folkerts and Saskia Braber

Cells 2022, 11(18), 2893; https://doi.org/10.3390/cells11182893 - 16 Sep 2022

Cited by 16 | Viewed by 5837

Abstract

Chronic obstructive pulmonary disease (COPD), often caused by smoking, is a chronic lung disease with systemic manifestations including metabolic comorbidities. This study investigates adaptive and pathological alterations in adipose and skeletal muscle tissue following cigarette smoke exposure using in vivo and in vitro [...] Read more.

Chronic obstructive pulmonary disease (COPD), often caused by smoking, is a chronic lung disease with systemic manifestations including metabolic comorbidities. This study investigates adaptive and pathological alterations in adipose and skeletal muscle tissue following cigarette smoke exposure using in vivo and in vitro models. Mice were exposed to cigarette smoke or air for 72 days and the pre-adipose cell line 3T3-L1 was utilized as an in vitro model. Cigarette smoke exposure decreased body weight, and the proportional loss in fat mass was more pronounced than the lean mass loss. Cigarette smoke exposure reduced adipocyte size and increased adipocyte numbers. Adipose macrophage numbers and associated cytokine levels, including interleukin-1β, interleukine-6 and tumor necrosis factor-α were elevated in smoke-exposed mice. Muscle strength and protein synthesis signaling were decreased after smoke exposure; however, muscle mass was not changed. In vitro studies demonstrated that lipolysis and fatty acid oxidation were upregulated in cigarette smoke-exposed pre-adipocytes. In conclusion, cigarette smoke exposure induces a loss of whole-body fat mass and adipose atrophy, which is likely due to enhanced lipolysis. Full article

(This article belongs to the Special Issue Metabolic Alterations and Cellular Stress Responses in Chronic Obstructive Pulmonary Disease)

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15 pages, 3542 KiB

Open AccessEditor’s ChoiceArticle

Characterization of Three-Dimensional Trophoblast Spheroids: An Alternative Model to Study the Physiological Properties of the Placental Unit

by Violeta Stojanovska, Susanne Arnold, Mario Bauer, Hermann Voss, Stefan Fest and Ana Claudia Zenclussen

Cells 2022, 11(18), 2884; https://doi.org/10.3390/cells11182884 - 15 Sep 2022

Cited by 17 | Viewed by 4125

Abstract

It was postulated that 3D cell culture models more accurately reflect the complex tissue physiology and morphology in comparison to 2D cell monolayers. Currently, there is a shortage of well-characterized and easily maintainable high-throughput experimental models of the human placenta. Here, we characterized [...] Read more.

It was postulated that 3D cell culture models more accurately reflect the complex tissue physiology and morphology in comparison to 2D cell monolayers. Currently, there is a shortage of well-characterized and easily maintainable high-throughput experimental models of the human placenta. Here, we characterized three different 3D cultures (e.g., spheroids) derived from trophoblast cell lines and studied their functionality in comparison to primary fetal trophoblasts and placental tissue. The spheroid growth rates of JEG3, BeWo and HTR8/SVneo cell lines were similar among each other and were significantly larger in comparison to primary trophoblast spheroids. All spheroids exhibited migratory properties and shortest distances were registered for JEG3 spheroids. Even though all spheroids displayed invasive capabilities, only the invasive features of HTR8/SVneo spheroids resulted in specific branching. This was in agreement with the invasive properties of the spheroids obtained from primary trophoblasts. Human chorionic gonadotropin production was highest in JEG3 spheroids and only increased when stimulated with cAMP and forskolin in BeWo, but not HTR8/SVneo spheroids. The gene expression analysis confirmed that 3D trophoblast cell cultures and especially HTR8/SVneo spheroids showed considerable similarities with the gene expression profile of primary placental tissue. This study offers a broad characterization of 3D trophoblast spheroids that, in turn, can help in selecting the best model depending on the scientific question that needs to be answered. Full article

(This article belongs to the Special Issue Placental Development in Health and Disease)

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23 pages, 4047 KiB

Open AccessEditor’s ChoiceArticle

The ABA-LANCL1/2 Hormone-Receptors System Protects H9c2 Cardiomyocytes from Hypoxia-Induced Mitochondrial Injury via an AMPK- and NO-Mediated Mechanism

by Sonia Spinelli, Lucrezia Guida, Tiziana Vigliarolo, Mario Passalacqua, Giulia Begani, Mirko Magnone, Laura Sturla, Andrea Benzi, Pietro Ameri, Edoardo Lazzarini, Claudia Bearzi, Roberto Rizzi and Elena Zocchi

Cells 2022, 11(18), 2888; https://doi.org/10.3390/cells11182888 - 15 Sep 2022

Cited by 19 | Viewed by 4326

Abstract

Abscisic acid (ABA) regulates plant responses to stress, partly via NO. In mammals, ABA stimulates NO production by innate immune cells and keratinocytes, glucose uptake and mitochondrial respiration by skeletal myocytes and improves blood glucose homeostasis through its receptors LANCL1 and LANCL2. We [...] Read more.

Abscisic acid (ABA) regulates plant responses to stress, partly via NO. In mammals, ABA stimulates NO production by innate immune cells and keratinocytes, glucose uptake and mitochondrial respiration by skeletal myocytes and improves blood glucose homeostasis through its receptors LANCL1 and LANCL2. We hypothesized a role for the ABA-LANCL1/2 system in cardiomyocyte protection from hypoxia via NO. The effect of ABA and of the silencing or overexpression of LANCL1 and LANCL2 were investigated in H9c2 rat cardiomyoblasts under normoxia or hypoxia/reoxygenation. In H9c2, hypoxia induced ABA release, and ABA stimulated NO production. ABA increased the survival of H9c2 to hypoxia, and L-NAME, an inhibitor of NO synthase (NOS), abrogated this effect. ABA also increased glucose uptake and NADPH levels and increased phosphorylation of Akt, AMPK and eNOS. Overexpression or silencing of LANCL1/2 significantly increased or decreased, respectively, transcription, expression and phosphorylation of AMPK, Akt and eNOS; transcription of NAMPT, Sirt1 and the arginine transporter. The mitochondrial proton gradient and cell vitality increased in LANCL1/2-overexpressing vs. -silenced cells after hypoxia/reoxygenation, and L-NAME abrogated this difference. These results implicate the ABA-LANCL1/2 hormone-receptor system in NO-mediated cardiomyocyte protection against hypoxia. Full article

(This article belongs to the Collection Regulation of Cell Function by AMPK and Sirtuins: From Basic Research to Disease and Aging)

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14 pages, 2204 KiB

Open AccessEditor’s ChoiceArticle

Biological Pathway-Derived TMB Robustly Predicts the Outcome of Immune Checkpoint Blockade Therapy

by Ya-Ru Miao, Chun-Jie Liu, Hui Hu, Mei Yang and An-Yuan Guo

Cells 2022, 11(18), 2802; https://doi.org/10.3390/cells11182802 - 8 Sep 2022

Cited by 7 | Viewed by 3173

Abstract

Although immune checkpoint blockade (ICB) therapies have achieved great progress, the patient response varies among cancers. In this study, we analyzed the potential genomic indicators contributing to ICB therapy response. The results showed that high tumor mutation burden (TMB) failed to predict response [...] Read more.

Although immune checkpoint blockade (ICB) therapies have achieved great progress, the patient response varies among cancers. In this study, we analyzed the potential genomic indicators contributing to ICB therapy response. The results showed that high tumor mutation burden (TMB) failed to predict response in anti-PD1 treated melanoma. SERPINB3 was the most significant response-related gene in melanoma and mutations in either SERPINB3 or PEG3 can serve as an independent risk factor in melanoma. Some recurrent mutations in CSMD3 were only in responders or non-responders, indicating their diverse impacts on patient response. Enrichment scores (ES) of gene mutations in 12 biological pathways were significantly higher in responders or non-responders. Next, the P-TMB calculated from genes in these pathways was significantly related to patient response with prediction AUC 0.74–0.82 in all collected datasets. In conclusion, our work provides new insights into the application of TMB in predicting patient response, which will benefit to immunotherapy research. Full article

(This article belongs to the Special Issue Cutting Edge in Bioinformatics of Cancer Immunotherapy)

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16 pages, 1852 KiB

Open AccessEditor’s ChoiceArticle

BC-miR: Monitoring Breast Cancer-Related miRNA Profile in Blood Sera—A Prosperous Approach for Tumor Detection

by Barbara N. Borsos, Zoltán G. Páhi, Zsuzsanna Ujfaludi, Farkas Sükösd, Alíz Nikolényi, Sarolta Bankó, Gabriella Pankotai-Bodó, Orsolya Oláh-Németh and Tibor Pankotai

Cells 2022, 11(17), 2721; https://doi.org/10.3390/cells11172721 - 31 Aug 2022

Cited by 14 | Viewed by 3224

Abstract

Breast cancer is the most frequent cancer with a high fatality rate amongst women worldwide. Diagnosing at an early stage is challenging, and due to the limitations of the currently used techniques, including mammography and imaging diagnostics, it still remains unascertained. Serum biomarkers [...] Read more.

Breast cancer is the most frequent cancer with a high fatality rate amongst women worldwide. Diagnosing at an early stage is challenging, and due to the limitations of the currently used techniques, including mammography and imaging diagnostics, it still remains unascertained. Serum biomarkers can be a solution for this as they can be isolated in a less painful, more cost-effective, and minimally invasive manner. In this study, we shed light on the relevant role of multiple microRNAs (miRNAs) as potential biomarkers in breast cancer diagnosis. We monitored the expressional changes of 15 pre-selected miRNAs in a large cohort, including 65 patients with breast cancer and 42 healthy individuals. We performed thorough statistical analyses on the cohort sample set and determined the diagnostic accuracy of individual and multiple miRNAs. Our study reveals a potential improvement in diagnostics by implicating the monitoring of miR-15a+miR-16+miR-221 expression in breast cancer management. Full article

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25 pages, 6296 KiB

Open AccessEditor’s ChoiceArticle

The Proteome Signatures of Fibroblasts from Patients with Severe, Intermediate and Mild Spinal Muscular Atrophy Show Limited Overlap

by Sharon J. Brown, Rachel A. Kline, Silvia A. Synowsky, Sally L. Shirran, Ian Holt, Kelly A. Sillence, Peter Claus, Brunhilde Wirth, Thomas M. Wishart and Heidi R. Fuller

Cells 2022, 11(17), 2624; https://doi.org/10.3390/cells11172624 - 23 Aug 2022

Cited by 7 | Viewed by 4175

Abstract

Most research to characterise the molecular consequences of spinal muscular atrophy (SMA) has focused on SMA I. Here, proteomic profiling of skin fibroblasts from severe (SMA I), intermediate (SMA II), and mild (SMA III) patients, alongside age-matched controls, was conducted using SWATH mass [...] Read more.

Most research to characterise the molecular consequences of spinal muscular atrophy (SMA) has focused on SMA I. Here, proteomic profiling of skin fibroblasts from severe (SMA I), intermediate (SMA II), and mild (SMA III) patients, alongside age-matched controls, was conducted using SWATH mass spectrometry analysis. Differentially expressed proteomic profiles showed limited overlap across each SMA type, and variability was greatest within SMA II fibroblasts, which was not explained by SMN2 copy number. Despite limited proteomic overlap, enriched canonical pathways common to two of three SMA severities with at least one differentially expressed protein from the third included mTOR signalling, regulation of eIF2 and eIF4 signalling, and protein ubiquitination. Network expression clustering analysis identified protein profiles that may discriminate or correlate with SMA severity. From these clusters, the differential expression of PYGB (SMA I), RAB3B (SMA II), and IMP1 and STAT1 (SMA III) was verified by Western blot. All SMA fibroblasts were transfected with an SMN-enhanced construct, but only RAB3B expression in SMA II fibroblasts demonstrated an SMN-dependent response. The diverse proteomic profiles and pathways identified here pave the way for studies to determine their utility as biomarkers for patient stratification or monitoring treatment efficacy and for the identification of severity-specific treatments. Full article

(This article belongs to the Special Issue Proteomic Applications in Ageing and Neurodegenerative Conditions)

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15 pages, 2191 KiB

Open AccessEditor’s ChoiceArticle

A Multi-Strain Probiotic Formulation Improves Intestinal Barrier Function by the Modulation of Tight and Adherent Junction Proteins

by Raffaella di Vito, Carmela Conte and Giovanna Traina

Cells 2022, 11(16), 2617; https://doi.org/10.3390/cells11162617 - 22 Aug 2022

Cited by 45 | Viewed by 7726

Abstract

In healthy individuals, tight junction proteins (TJPs) maintain the integrity of the intestinal barrier. Dysbiosis and increased intestinal permeability are observed in several diseases, such as inflammatory bowel disease. Many studies highlight the role of probiotics in preventing intestinal barrier dysfunction. The present [...] Read more.

In healthy individuals, tight junction proteins (TJPs) maintain the integrity of the intestinal barrier. Dysbiosis and increased intestinal permeability are observed in several diseases, such as inflammatory bowel disease. Many studies highlight the role of probiotics in preventing intestinal barrier dysfunction. The present study aims to investigate the effects of a commercially available probiotic formulation of L. rhamnosus LR 32, B. lactis BL 04, and B. longum BB 536 (Serobioma, Bromatech s.r.l., Milan, Italy) on TJPs and the integrity of the intestinal epithelial barrier, and the ability of this formulation to prevent lipopolysaccharide-induced, inflammation-associated damage. An in vitro model of the intestinal barrier was developed using a Caco-2 cell monolayer. The mRNA expression levels of the TJ genes were analyzed using real-time PCR. Changes in the amounts of proteins were assessed with Western blotting. The effect of Serobioma on the intestinal epithelial barrier function was assessed using transepithelial electrical resistance (TEER) measurements. The probiotic formulation tested in this study modulates the expression of TJPs and prevents inflammatory damage. Our findings provide new insights into the mechanisms by which probiotics are able to prevent damage to the gut epithelial barrier. Full article

(This article belongs to the Special Issue Gut Microbiota in Nutrition and Health)

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18 pages, 5314 KiB

Open AccessEditor’s ChoiceArticle

Pathogenetic Mechanisms Underlying Spinocerebellar Ataxia Type 3 Are Altered in Primary Oligodendrocyte Culture

by Kristen H. Schuster, Alexandra F. Putka and Hayley S. McLoughlin

Cells 2022, 11(16), 2615; https://doi.org/10.3390/cells11162615 - 22 Aug 2022

Cited by 8 | Viewed by 3592

Abstract

Emerging evidence has implicated non-neuronal cells, particularly oligodendrocytes, in the pathophysiology of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease and Spinocerebellar ataxia type 3 (SCA3). We recently demonstrated that cell-autonomous dysfunction of oligodendrocyte maturation is one of [...] Read more.

Emerging evidence has implicated non-neuronal cells, particularly oligodendrocytes, in the pathophysiology of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease and Spinocerebellar ataxia type 3 (SCA3). We recently demonstrated that cell-autonomous dysfunction of oligodendrocyte maturation is one of the of the earliest and most robust changes in vulnerable regions of the SCA3 mouse brain. However, the cell- and disease-specific mechanisms that underlie oligodendrocyte dysfunction remain poorly understood and are difficult to isolate in vivo. In this study, we used primary oligodendrocyte cultures to determine how known pathogenic SCA3 mechanisms affect this cell type. We isolated oligodendrocyte progenitor cells from 5- to 7-day-old mice that overexpress human mutant ATXN3 or lack mouse ATXN3 and differentiated them for up to 5 days in vitro. Utilizing immunocytochemistry, we characterized the contributions of ATXN3 toxic gain-of-function and loss-of-function in oligodendrocyte maturation, protein quality pathways, DNA damage signaling, and methylation status. We illustrate the utility of primary oligodendrocyte culture for elucidating cell-specific pathway dysregulation relevant to SCA3. Given recent work demonstrating disease-associated oligodendrocyte signatures in other neurodegenerative diseases, this novel model has broad applicability in revealing mechanistic insights of oligodendrocyte contribution to pathogenesis. Full article

(This article belongs to the Collection Spinocerebellar Ataxia (SCA): Molecular Mechanisms and Novel Treatment Strategies)

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32 pages, 5494 KiB

Open AccessEditor’s ChoiceArticle

Single Cell Analysis of Cultivated Fibroblasts from Chronic Pancreatitis and Pancreatic Cancer Patients

by Yoshiaki Sunami, Yijun Chen, Bogusz Trojanowicz, Matthias Sommerer, Monika Hämmerle, Roland Eils and Jörg Kleeff

Cells 2022, 11(16), 2583; https://doi.org/10.3390/cells11162583 - 19 Aug 2022

Cited by 5 | Viewed by 4391

Abstract

Cancer-associated fibroblasts (CAFs) play a major role in the progression and drug resistance of pancreatic cancer. Recent studies suggest that CAFs exhibit functional heterogeneity and distinct transcriptomic signatures in pancreatic cancer. Pancreatic fibroblasts also form an integral component in pancreatic diseases such as [...] Read more.

Cancer-associated fibroblasts (CAFs) play a major role in the progression and drug resistance of pancreatic cancer. Recent studies suggest that CAFs exhibit functional heterogeneity and distinct transcriptomic signatures in pancreatic cancer. Pancreatic fibroblasts also form an integral component in pancreatic diseases such as chronic pancreatitis named disease-associated fibroblasts (DAFs). However, intra-tumoral heterogeneity of CAFs in pancreatic cancer patients and their pivotal role in cancer-related mechanisms have not been fully elucidated. Further, it has not been elucidated whether CAF subtypes identified in pancreatic cancer also exist in chronic pancreatitis. In this study, we used primary isolated fibroblasts from pancreatic cancer and chronic pancreatitis patients using the outgrowth method. Single-cell RNA sequencing (scRNA-seq) was performed, and bioinformatics analysis identified highly variable genes, including factors associated with overall survival of pancreatic cancer patients. The majority of highly variable genes are involved in the cell cycle. Instead of previously classified myofibroblastic (myCAFs), inflammatory (iCAFs), and antigen-presenting (ap) CAFs, we identified a myCAFs-like subtype in all cases. Most interestingly, after cell cycle regression, we observed 135 highly variable genes commonly identified in chronic pancreatitis and pancreatic cancer patients. This study is the first to conduct scRNAseq and bioinformatics analyses to compare CAFs/DAFs from both chronic pancreatitis and pancreatic cancer patients. Further studies are required to select and identify stromal factors in DAFs from chronic pancreatitis cases, which are commonly expressed also in CAFs potentially contributing to pancreatic cancer development. Full article

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13 pages, 2934 KiB

Open AccessEditor’s ChoiceArticle

Oxidative Stress Is Associated with Overgrowth in Drosophila l(3)mbt Mutant Imaginal Discs

by Paula Climent-Cantó, Cristina Molnar, Paula Santabárbara-Ruiz, Cristina Prieto, Josep F. Abril, Florenci Serras and Cayetano Gonzalez

Cells 2022, 11(16), 2542; https://doi.org/10.3390/cells11162542 - 16 Aug 2022

Cited by 1 | Viewed by 2820

Abstract

The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 °C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report [...] Read more.

The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 °C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report the results of a study of the hyperplastic l(3)mbt mutant wing imaginal discs. We identify the l(3)mbt wing disc tumour transcriptome and find it to include genes involved in reactive oxygen species (ROS) metabolism. Furthermore, we show the presence of oxidative stress in l(3)mbt hyperplastic discs, even in apoptosis-blocked conditions, but not in l(3)mbt brain tumours. We also find that chemically blocking oxidative stress in l(3)mbt wing discs reduces the incidence of wing disc overgrowths. Our results reveal the involvement of oxidative stress in l(3)mbt wing discs hyperplastic growth. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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13 pages, 2474 KiB

Open AccessEditor’s ChoiceArticle

Integrated Multi-Omics Signature Predicts Survival in Head and Neck Cancer

by Ilda Patrícia Ribeiro, Luísa Esteves, Francisco Caramelo, Isabel Marques Carreira and Joana Barbosa Melo

Cells 2022, 11(16), 2536; https://doi.org/10.3390/cells11162536 - 16 Aug 2022

Cited by 11 | Viewed by 2665

Abstract

Head and Neck Cancer (HNC) is characterized by phenotypic, biological, and clinical heterogeneity. Despite treatment modalities, approximately half of all patients will die of the disease. Several molecular biomarkers have been investigated, but until now, without clinical translation. Here, we identified an integrative [...] Read more.

Head and Neck Cancer (HNC) is characterized by phenotypic, biological, and clinical heterogeneity. Despite treatment modalities, approximately half of all patients will die of the disease. Several molecular biomarkers have been investigated, but until now, without clinical translation. Here, we identified an integrative nine-gene multi-omics signature correlated with HNC patients’ survival independently of relapses or metastasis development. This prognosis multi-omic signature comprises genes mapped in the chromosomes 1q, 3p, 8q, 17q, 19p, and 19q and encompasses alterations at copy number, gene expression, and methylation. Copy number alterations in LMCD1-A1S and GRM7, the methylation status of CEACAM19, KRT17, and ST18, and the expression profile of RPL29, UBA7, FCGR2C, and RPSAP58 can predict the HNC patients’ survival. The difference higher than two years observed in the survival of HNC patients that harbor this nine-gene multi-omics signature can represent a significant step forward to improve patients’ management and guide new therapeutic targets development. Full article

(This article belongs to the Special Issue Early Biomarkers of Cancer: Diagnosis and Progression)

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16 pages, 3108 KiB

Open AccessEditor’s ChoiceArticle

Integration of Human Protein Sequence and Protein-Protein Interaction Data by Graph Autoencoder to Identify Novel Protein-Abnormal Phenotype Associations

by Yuan Liu, Ruirui He, Yingjie Qu, Yuan Zhu, Dianke Li, Xinping Ling, Simin Xia, Zhenqiu Li and Dong Li

Cells 2022, 11(16), 2485; https://doi.org/10.3390/cells11162485 - 10 Aug 2022

Cited by 5 | Viewed by 3265

Abstract

Understanding gene functions and their associated abnormal phenotypes is crucial in the prevention, diagnosis and treatment against diseases. The Human Phenotype Ontology (HPO) is a standardized vocabulary for describing the phenotype abnormalities associated with human diseases. However, the current HPO annotations are far [...] Read more.

Understanding gene functions and their associated abnormal phenotypes is crucial in the prevention, diagnosis and treatment against diseases. The Human Phenotype Ontology (HPO) is a standardized vocabulary for describing the phenotype abnormalities associated with human diseases. However, the current HPO annotations are far from completion, and only a small fraction of human protein-coding genes has HPO annotations. Thus, it is necessary to predict protein-phenotype associations using computational methods. Protein sequences can indicate the structure and function of the proteins, and interacting proteins are more likely to have same function. It is promising to integrate these features for predicting HPO annotations of human protein. We developed GraphPheno, a semi-supervised method based on graph autoencoders, which does not require feature engineering to capture deep features from protein sequences, while also taking into account the topological properties in the protein–protein interaction network to predict the relationships between human genes/proteins and abnormal phenotypes. Cross validation and independent dataset tests show that GraphPheno has satisfactory prediction performance. The algorithm is further confirmed on automatic HPO annotation for no-knowledge proteins under the benchmark of the second Critical Assessment of Functional Annotation, 2013–2014 (CAFA2), where GraphPheno surpasses most existing methods. Further bioinformatics analysis shows that predicted certain phenotype-associated genes using GraphPheno share similar biological properties with known ones. In a case study on the phenotype of abnormality of mitochondrial respiratory chain, top prioritized genes are validated by recent papers. We believe that GraphPheno will help to reveal more associations between genes and phenotypes, and contribute to the discovery of drug targets. Full article

(This article belongs to the Special Issue New Insights into Proteomics and Post-translational Modification of Proteins)

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