Cells

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 1105

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 2023)

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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 1044

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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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

Viewed by 1119

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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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 1200

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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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 1 | Viewed by 1081

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

Viewed by 894

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 1249

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 1146

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

Viewed by 1833

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

Viewed by 1283

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 847

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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23 pages, 398 KiB

Open AccessEditor’s ChoiceReview

The Genetic Basis of Non-Contact Soft Tissue Injuries-Are There Practical Applications of Genetic Knowledge?

by Beata Borzemska, Paweł Cięszczyk and Cezary Żekanowski

Cells 2024, 13(22), 1828; https://doi.org/10.3390/cells13221828 - 5 Nov 2024

Viewed by 1723

Abstract

Physical activity increases the risk of non-contact injuries, mainly affecting muscles, tendons, and ligaments. Genetic factors are recognized as contributing to susceptibility to different types of soft tissue injuries, making this broad condition a complicated multifactorial entity. Understanding genetic predisposition seems to offer [...] Read more.

Physical activity increases the risk of non-contact injuries, mainly affecting muscles, tendons, and ligaments. Genetic factors are recognized as contributing to susceptibility to different types of soft tissue injuries, making this broad condition a complicated multifactorial entity. Understanding genetic predisposition seems to offer the potential for personalized injury prevention and improved recovery strategies. The candidate gene analysis approach used so far, has often yielded inconclusive results. This manuscript reviews the most commonly studied genetic variants in genes involved in the musculoskeletal system’s structure and recovery processes (ACTN3, ACE, CKM, MLCK, AMPD1, IGF2, IL6, TNFα, CCL2, COL1A1, COL5A1, MMP3, and TNC). Referring to the literature, it was highlighted that single-gene analyses provide limited insight. On the other hand, novel genetic testing methods identify numerous variants of uncertain physiological relevance. Distinguishing between functionally important variants, modifying variants, and the thousands of irrelevant variants requires advanced bioinformatics methods and basic multiomics research to identify the key biological pathways contributing to injury susceptibility. Tools like the Total Genotype Score (TGS) and Polygenic Risk Score (PRS) offer a more holistic view by assessing the combined effect of multiple variants. However, these methods, while useful in research, lack clinical applicability. In conclusion, it is too early to determine the clinical implications of genetic variability as a tool for improving well-established training and injury prevention methods, as the predictive power of genetic testing for injury predisposition is currently low. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Musculoskeletal and Other Tissue Injuries and Disruptions—Regenerative Potential of Exercise)

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 1 | Viewed by 1040

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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24 pages, 2902 KiB

Open AccessEditor’s ChoiceReview

Advancing CRISPR-Based Solutions for COVID-19 Diagnosis and Therapeutics

by Roaa Hadi, Abhishek Poddar, Shivakumar Sonnaila, Venkata Suryanarayana Murthy Bhavaraju and Shilpi Agrawal

Cells 2024, 13(21), 1794; https://doi.org/10.3390/cells13211794 - 30 Oct 2024

Cited by 1 | Viewed by 2558

Abstract

Since the onset of the COVID-19 pandemic, a variety of diagnostic approaches, including RT-qPCR, RAPID, and LFA, have been adopted, with RT-qPCR emerging as the gold standard. However, a significant challenge in COVID-19 diagnostics is the wide range of symptoms presented by patients, [...] Read more.

Since the onset of the COVID-19 pandemic, a variety of diagnostic approaches, including RT-qPCR, RAPID, and LFA, have been adopted, with RT-qPCR emerging as the gold standard. However, a significant challenge in COVID-19 diagnostics is the wide range of symptoms presented by patients, necessitating early and accurate diagnosis for effective management. Although RT-qPCR is a precise molecular technique, it is not immune to false-negative results. In contrast, CRISPR-based detection methods for SARS-CoV-2 offer several advantages: they are cost-effective, time-efficient, highly sensitive, and specific, and they do not require sophisticated instruments. These methods also show promise for scalability, enabling diagnostic tests. CRISPR technology can be customized to target any genomic region of interest, making it a versatile tool with applications beyond diagnostics, including therapeutic development. The CRISPR/Cas systems provide precise gene targeting with immense potential for creating next-generation diagnostics and therapeutics. One of the key advantages of CRISPR/Cas-based therapeutics is the ability to perform multiplexing, where different sgRNAs or crRNAs can target multiple sites within the same gene, reducing the likelihood of viral escape mutants. Among the various CRISPR systems, CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) are particularly promising. These systems can target a broad range of single-stranded RNA viruses, making them suitable for the diagnosis and treatment of various viral diseases, including SARS-CoV-2. However, the efficacy and safety of CRISPR-based therapeutics must be thoroughly evaluated in pre-clinical and clinical settings. While CRISPR biotechnologies have not yet been fully harnessed to control the current COVID-19 pandemic, there is an optimism that the limitations of the CRISPR/Cas system can be overcome soon. This review discusses how CRISPR-based strategies can revolutionize disease diagnosis and therapeutic development, better preparing us for future viral threats. Full article

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19 pages, 759 KiB

Open AccessEditor’s ChoiceReview

Hepatocellular-Carcinoma-Derived Organoids: Innovation in Cancer Research

by Carlo Airola, Maria Pallozzi, Eleonora Cesari, Lucia Cerrito, Leonardo Stella, Claudio Sette, Felice Giuliante, Antonio Gasbarrini and Francesca Romana Ponziani

Cells 2024, 13(20), 1726; https://doi.org/10.3390/cells13201726 - 18 Oct 2024

Cited by 3 | Viewed by 2267

Abstract

Hepatocellular carcinomas (HCCs) are highly heterogeneous malignancies. They are characterized by a peculiar tumor microenvironment and dense vascularization. The importance of signaling between immune cells, endothelial cells, and tumor cells leads to the difficult recapitulation of a reliable in vitro HCC model using [...] Read more.

Hepatocellular carcinomas (HCCs) are highly heterogeneous malignancies. They are characterized by a peculiar tumor microenvironment and dense vascularization. The importance of signaling between immune cells, endothelial cells, and tumor cells leads to the difficult recapitulation of a reliable in vitro HCC model using the conventional two-dimensional cell cultures. The advent of three-dimensional organoid tumor technology has revolutionized our understanding of the pathogenesis and progression of several malignancies by faithfully replicating the original cancer genomic, epigenomic, and microenvironmental landscape. Organoids more closely mimic the in vivo environment and cell interactions, replicating factors such as the spatial organization of cell surface receptors and gene expression, and will probably become an important tool in the choice of therapies and the evaluation of tumor response to treatments. This review aimed to describe the ongoing and potential applications of organoids as an in vitro model for the study of HCC development, its interaction with the host’s immunity, the analysis of drug sensitivity tests, and the current limits in this field. Full article

(This article belongs to the Special Issue Liver Constituent Cells: Their Niche, Close Intercellular Relationship and Crosstalk with the Extracellular Environment—Current and Future Perspectives (2nd Edition))

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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

Viewed by 7196

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

Viewed by 1603

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 2718

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 1546

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 1 | Viewed by 4454

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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21 pages, 6630 KiB

Open AccessEditor’s ChoiceReview

Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various In Vitro Disease Models: A Narrow Review

by Hiroshi Ohguro, Megumi Watanabe, Tatsuya Sato, Nami Nishikiori, Araya Umetsu, Megumi Higashide, Toshiyuki Yano, Hiromu Suzuki, Akihiro Miyazaki, Kohichi Takada, Hisashi Uhara, Masato Furuhashi and Fumihito Hikage

Cells 2024, 13(18), 1549; https://doi.org/10.3390/cells13181549 - 14 Sep 2024

Cited by 5 | Viewed by 2236

Abstract

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various [...] Read more.

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases. Full article

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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 3 | Viewed by 1881

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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21 pages, 1753 KiB

Open AccessEditor’s ChoiceReview

V-ATPase Dysfunction in the Brain: Genetic Insights and Therapeutic Opportunities

by Antonio Falace, Greta Volpedo, Marcello Scala, Federico Zara, Pasquale Striano and Anna Fassio

Cells 2024, 13(17), 1441; https://doi.org/10.3390/cells13171441 - 28 Aug 2024

Cited by 3 | Viewed by 2818

Abstract

Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H⁺ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels [...] Read more.

Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H⁺ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels in the brain and its impairment triggers neuronal dysfunction and neurodegeneration. Due to their post-mitotic nature and highly specialized function and morphology, neurons display a unique vulnerability to lysosomal dyshomeostasis. Alterations in genes encoding subunits composing v-ATPase or v-ATPase-related proteins impair brain development and synaptic function in animal models and underlie genetic diseases in humans, such as encephalopathies, epilepsy, as well as neurodevelopmental, and degenerative disorders. This review presents the genetic and functional evidence linking v-ATPase subunits and accessory proteins to various brain disorders, from early-onset developmental epileptic encephalopathy to neurodegenerative diseases. We highlight the latest emerging therapeutic strategies aimed at mitigating lysosomal defects associated with v-ATPase dysfunction. Full article

(This article belongs to the Special Issue Understanding the Interplay Between Autophagy and Neurodegeneration)

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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 2 | Viewed by 1705

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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29 pages, 2202 KiB

Open AccessEditor’s ChoiceReview

Anti-Inflammatory Role of the Klotho Protein and Relevance to Aging

by Gérald J. Prud’homme and Qinghua Wang

Cells 2024, 13(17), 1413; https://doi.org/10.3390/cells13171413 - 24 Aug 2024

Cited by 9 | Viewed by 5854

Abstract

The α-Klotho protein (hereafter Klotho) is an obligate coreceptor for fibroblast growth factor 23 (FGF23). It is produced in the kidneys, brain and other sites. Klotho insufficiency causes hyperphosphatemia and other anomalies. Importantly, it is associated with chronic pathologies (often age-related) that have [...] Read more.

The α-Klotho protein (hereafter Klotho) is an obligate coreceptor for fibroblast growth factor 23 (FGF23). It is produced in the kidneys, brain and other sites. Klotho insufficiency causes hyperphosphatemia and other anomalies. Importantly, it is associated with chronic pathologies (often age-related) that have an inflammatory component. This includes atherosclerosis, diabetes and Alzheimer’s disease. Its mode of action in these diseases is not well understood, but it inhibits or regulates multiple major pathways. Klotho has a membrane form and a soluble form (s-Klotho). Cytosolic Klotho is postulated but not well characterized. s-Klotho has endocrine properties that are incompletely elucidated. It binds to the FGF receptor 1c (FGFR1c) that is widely expressed (including endothelial cells). It also attaches to soluble FGF23, and FGF23/Klotho binds to FGFRs. Thus, s-Klotho might be a roaming FGF23 coreceptor, but it has other functions. Notably, Klotho (cell-bound or soluble) counteracts inflammation and appears to mitigate related aging (inflammaging). It inhibits NF-κB and the NLRP3 inflammasome. This inflammasome requires priming by NF-κB and produces active IL-1β, membrane pores and cell death (pyroptosis). In accord, Klotho countered inflammation and cell injury induced by toxins, damage-associated molecular patterns (DAMPs), cytokines, and reactive oxygen species (ROS). s-Klotho also blocks the TGF-β receptor and Wnt ligands, which lessens fibrotic disease. Low Klotho is associated with loss of muscle mass (sarcopenia), as occurs in aging and chronic diseases. s-Klotho counters the inhibitory effects of myostatin and TGF-β on muscle, reduces inflammation, and improves muscle repair following injury. The inhibition of TGF-β and other factors may also be protective in diabetic retinopathy and age-related macular degeneration (AMD). This review examines Klotho functions especially as related to inflammation and potential applications. Full article

(This article belongs to the Section Tissues and Organs)

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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 2 | Viewed by 1531

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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16 pages, 2568 KiB

Open AccessEditor’s ChoiceReview

Molecular Mechanisms of Autophagy Decline during Aging

by Shaun H. Y. Lim, Malene Hansen and Caroline Kumsta

Cells 2024, 13(16), 1364; https://doi.org/10.3390/cells13161364 - 16 Aug 2024

Cited by 5 | Viewed by 4348

Abstract

Macroautophagy (hereafter autophagy) is a cellular recycling process that degrades cytoplasmic components, such as protein aggregates and mitochondria, and is associated with longevity and health in multiple organisms. While mounting evidence supports that autophagy declines with age, the underlying molecular mechanisms remain unclear. [...] Read more.

Macroautophagy (hereafter autophagy) is a cellular recycling process that degrades cytoplasmic components, such as protein aggregates and mitochondria, and is associated with longevity and health in multiple organisms. While mounting evidence supports that autophagy declines with age, the underlying molecular mechanisms remain unclear. Since autophagy is a complex, multistep process, orchestrated by more than 40 autophagy-related proteins with tissue-specific expression patterns and context-dependent regulation, it is challenging to determine how autophagy fails with age. In this review, we describe the individual steps of the autophagy process and summarize the age-dependent molecular changes reported to occur in specific steps of the pathway that could impact autophagy. Moreover, we describe how genetic manipulations of autophagy-related genes can affect lifespan and healthspan through studies in model organisms and age-related disease models. Understanding the age-related changes in each step of the autophagy process may prove useful in developing approaches to prevent autophagy decline and help combat a number of age-related diseases with dysregulated autophagy. Full article

(This article belongs to the Special Issue Autophagy Meets Aging 2025)

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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 1513

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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16 pages, 1262 KiB

Open AccessEditor’s ChoiceReview

Importance of Autophagy Regulation in Glioblastoma with Temozolomide Resistance

by Young Keun Hwang, Dong-Hun Lee, Eun Chae Lee and Jae Sang Oh

Cells 2024, 13(16), 1332; https://doi.org/10.3390/cells13161332 - 11 Aug 2024

Cited by 2 | Viewed by 2621

Abstract

Glioblastoma (GBM) is the most aggressive and common malignant and CNS tumor, accounting for 47.7% of total cases. Glioblastoma has an incidence rate of 3.21 cases per 100,000 people. The regulation of autophagy, a conserved cellular process involved in the degradation and recycling [...] Read more.

Glioblastoma (GBM) is the most aggressive and common malignant and CNS tumor, accounting for 47.7% of total cases. Glioblastoma has an incidence rate of 3.21 cases per 100,000 people. The regulation of autophagy, a conserved cellular process involved in the degradation and recycling of cellular components, has been found to play an important role in GBM pathogenesis and response to therapy. Autophagy plays a dual role in promoting tumor survival and apoptosis, and here we discuss the complex interplay between autophagy and GBM. We summarize the mechanisms underlying autophagy dysregulation in GBM, including PI3K/AKT/mTOR signaling, which is most active in brain tumors, and EGFR and mutant EGFRvIII. We also review potential therapeutic strategies that target autophagy for the treatment of GBM, such as autophagy inhibitors used in combination with the standard of care, TMZ. We discuss our current understanding of how autophagy is involved in TMZ resistance and its role in glioblastoma development and survival. Full article

(This article belongs to the Section Autophagy)

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24 pages, 663 KiB

Open AccessEditor’s ChoiceReview

In Silico Deciphering of the Potential Impact of Variants of Uncertain Significance in Hereditary Colorectal Cancer Syndromes

by Candida Fasano, Martina Lepore Signorile, Katia De Marco, Giovanna Forte, Vittoria Disciglio, Paola Sanese, Valentina Grossi and Cristiano Simone

Cells 2024, 13(16), 1314; https://doi.org/10.3390/cells13161314 - 6 Aug 2024

Cited by 2 | Viewed by 1923

Abstract

Colorectal cancer (CRC) ranks third in terms of cancer incidence worldwide and is responsible for 8% of all deaths globally. Approximately 10% of CRC cases are caused by inherited pathogenic mutations in driver genes involved in pathways that are crucial for CRC tumorigenesis [...] Read more.

Colorectal cancer (CRC) ranks third in terms of cancer incidence worldwide and is responsible for 8% of all deaths globally. Approximately 10% of CRC cases are caused by inherited pathogenic mutations in driver genes involved in pathways that are crucial for CRC tumorigenesis and progression. These hereditary mutations significantly increase the risk of initial benign polyps or adenomas developing into cancer. In recent years, the rapid and accurate sequencing of CRC-specific multigene panels by next-generation sequencing (NGS) technologies has enabled the identification of several recurrent pathogenic variants with established functional consequences. In parallel, rare genetic variants that are not characterized and are, therefore, called variants of uncertain significance (VUSs) have also been detected. The classification of VUSs is a challenging task because each amino acid has specific biochemical properties and uniquely contributes to the structural stability and functional activity of proteins. In this scenario, the ability to computationally predict the effect of a VUS is crucial. In particular, in silico prediction methods can provide useful insights to assess the potential impact of a VUS and support additional clinical evaluation. This approach can further benefit from recent advances in artificial intelligence-based technologies. In this review, we describe the main in silico prediction tools that can be used to evaluate the structural and functional impact of VUSs and provide examples of their application in the analysis of gene variants involved in hereditary CRC syndromes. Full article

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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 3 | Viewed by 3805

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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32 pages, 1003 KiB

Open AccessEditor’s ChoiceReview

Molecular Mechanisms and Clinical Application of Multipotent Stem Cells for Spinal Cord Injury

by Michał Szymoniuk, Jakub Litak, Leon Sakwa, Aleksandra Dryla, Wojciech Zezuliński, Wojciech Czyżewski, Piotr Kamieniak and Tomasz Blicharski

Cells 2023, 12(1), 120; https://doi.org/10.3390/cells12010120 - 28 Dec 2022

Cited by 26 | Viewed by 6228

Abstract

Spinal Cord Injury (SCI) is a common neurological disorder with devastating psychical and psychosocial sequelae. The majority of patients after SCI suffer from permanent disability caused by motor dysfunction, impaired sensation, neuropathic pain, spasticity as well as urinary complications, and a small number [...] Read more.

Spinal Cord Injury (SCI) is a common neurological disorder with devastating psychical and psychosocial sequelae. The majority of patients after SCI suffer from permanent disability caused by motor dysfunction, impaired sensation, neuropathic pain, spasticity as well as urinary complications, and a small number of patients experience a complete recovery. Current standard treatment modalities of the SCI aim to prevent secondary injury and provide limited recovery of lost neurological functions. Stem Cell Therapy (SCT) represents an emerging treatment approach using the differentiation, paracrine, and self-renewal capabilities of stem cells to regenerate the injured spinal cord. To date, multipotent stem cells including mesenchymal stem cells (MSCs), neural stem cells (NSCs), and hematopoietic stem cells (HSCs) represent the most investigated types of stem cells for the treatment of SCI in preclinical and clinical studies. The microenvironment of SCI has a significant impact on the survival, proliferation, and differentiation of transplanted stem cells. Therefore, a deep understanding of the pathophysiology of SCI and molecular mechanisms through which stem cells act may help improve the treatment efficacy of SCT and find new therapeutic approaches such as stem-cell-derived exosomes, gene-modified stem cells, scaffolds, and nanomaterials. In this literature review, the pathogenesis of SCI and molecular mechanisms of action of multipotent stem cells including MSCs, NSCs, and HSCs are comprehensively described. Moreover, the clinical efficacy of multipotent stem cells in SCI treatment, an optimal protocol of stem cell administration, and recent therapeutic approaches based on or combined with SCT are also discussed. Full article

(This article belongs to the Special Issue Neural Repair of the Central Nervous System: Stem Cells and Other Approaches)

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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 6 | Viewed by 3178

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 7 | Viewed by 2822

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 2 | Viewed by 3952

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 2405

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 4 | Viewed by 3806

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 1 | Viewed by 3114

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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22 pages, 1310 KiB

Open AccessEditor’s ChoiceReview

The Interface of Tumour-Associated Macrophages with Dying Cancer Cells in Immuno-Oncology

by Isaure Vanmeerbeek, Jannes Govaerts, Raquel S. Laureano, Jenny Sprooten, Stefan Naulaerts, Daniel M. Borras, Damya Laoui, Massimiliano Mazzone, Jo A. Van Ginderachter and Abhishek D. Garg

Cells 2022, 11(23), 3890; https://doi.org/10.3390/cells11233890 - 2 Dec 2022

Cited by 17 | Viewed by 7053

Abstract

Tumour-associated macrophages (TAMs) are essential players in the tumour microenvironment (TME) and modulate various pro-tumorigenic functions such as immunosuppression, angiogenesis, cancer cell proliferation, invasion and metastasis, along with resistance to anti-cancer therapies. TAMs also mediate important anti-tumour functions and can clear dying cancer [...] Read more.

Tumour-associated macrophages (TAMs) are essential players in the tumour microenvironment (TME) and modulate various pro-tumorigenic functions such as immunosuppression, angiogenesis, cancer cell proliferation, invasion and metastasis, along with resistance to anti-cancer therapies. TAMs also mediate important anti-tumour functions and can clear dying cancer cells via efferocytosis. Thus, not surprisingly, TAMs exhibit heterogeneous activities and functional plasticity depending on the type and context of cancer cell death that they are faced with. This ultimately governs both the pro-tumorigenic and anti-tumorigenic activity of TAMs, making the interface between TAMs and dying cancer cells very important for modulating cancer growth and the efficacy of chemo-radiotherapy or immunotherapy. In this review, we discuss the interface of TAMs with cancer cell death from the perspectives of cell death pathways, TME-driven variations, TAM heterogeneity and cell-death-inducing anti-cancer therapies. We believe that a better understanding of how dying cancer cells influence TAMs can lead to improved combinatorial anti-cancer therapies, especially in combination with TAM-targeting immunotherapies. Full article

(This article belongs to the Special Issue Immunogenic Cell Stress and Death)

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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 2640

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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18 pages, 1837 KiB

Open AccessEditor’s ChoiceReview

A Review on Autophagy in Orofacial Neuropathic Pain

by Mayank Shrivastava and Liang Ye

Cells 2022, 11(23), 3842; https://doi.org/10.3390/cells11233842 - 30 Nov 2022

Cited by 5 | Viewed by 2828

Abstract

Orofacial neuropathic pain indicates pain caused by a lesion or diseases of the somatosensory nervous system. It is challenging for the clinician to diagnose and manage orofacial neuropathic pain conditions due to the considerable variability between individual clinical presentations and a lack of [...] Read more.

Orofacial neuropathic pain indicates pain caused by a lesion or diseases of the somatosensory nervous system. It is challenging for the clinician to diagnose and manage orofacial neuropathic pain conditions due to the considerable variability between individual clinical presentations and a lack of understanding of the mechanisms underlying the etiology and pathogenesis. In the last few decades, researchers have developed diagnostic criteria, questionnaires, and clinical assessment methods for the diagnosis of orofacial neuropathic pain. Recently, researchers have observed the role of autophagy in neuronal dysfunction as well as in the modulation of neuropathic pain. On this basis, in the present review, we highlight the characteristics, classification, and clinical assessment of orofacial neuropathic pain. Additionally, we introduce autophagy and its potential role in the modulation of orofacial neuropathic pain, along with a brief overview of the pathogenesis, which in future may reveal new possible targets for treating this condition. Full article

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

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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 2222

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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31 pages, 3152 KiB

Open AccessEditor’s ChoiceReview

Canonical and Noncanonical ER Stress-Mediated Autophagy Is a Bite the Bullet in View of Cancer Therapy

by Rashedul Alam, Mohammad Fazlul Kabir, Hyung-Ryong Kim and Han-Jung Chae

Cells 2022, 11(23), 3773; https://doi.org/10.3390/cells11233773 - 25 Nov 2022

Cited by 6 | Viewed by 4156

Abstract

Cancer cells adapt multiple mechanisms to counter intense stress on their way to growth. Tumor microenvironment stress leads to canonical and noncanonical endoplasmic stress (ER) responses, which mediate autophagy and are engaged during proteotoxic challenges to clear unfolded or misfolded proteins and damaged [...] Read more.

Cancer cells adapt multiple mechanisms to counter intense stress on their way to growth. Tumor microenvironment stress leads to canonical and noncanonical endoplasmic stress (ER) responses, which mediate autophagy and are engaged during proteotoxic challenges to clear unfolded or misfolded proteins and damaged organelles to mitigate stress. In these conditions, autophagy functions as a cytoprotective mechanism in which malignant tumor cells reuse degraded materials to generate energy under adverse growing conditions. However, cellular protection by autophagy is thought to be complicated, contentious, and context-dependent; the stress response to autophagy is suggested to support tumorigenesis and drug resistance, which must be adequately addressed. This review describes significant findings that suggest accelerated autophagy in cancer, a novel obstacle for anticancer therapy, and discusses the UPR components that have been suggested to be untreatable. Thus, addressing the UPR or noncanonical ER stress components is the most effective approach to suppressing cytoprotective autophagy for better and more effective cancer treatment. Full article

(This article belongs to the Special Issue Autophagy and Human Cancers)

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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 9 | Viewed by 3036

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 2802

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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42 pages, 1992 KiB

Open AccessFeature PaperEditor’s ChoiceReview

Generation of Lens Progenitor Cells and Lentoid Bodies from Pluripotent Stem Cells: Novel Tools for Human Lens Development and Ocular Disease Etiology

by Aleš Cvekl and Michael John Camerino

Cells 2022, 11(21), 3516; https://doi.org/10.3390/cells11213516 - 6 Nov 2022

Cited by 11 | Viewed by 4412

Abstract

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids [...] Read more.

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids and specific ocular tissues from pluripotent stem cells has provided invaluable insights into the formation of lineage-committed progenitor cell populations, signal transduction pathways, and self-organization principles. This review provides a comprehensive summary of recent advances in generation of adenohypophyseal, olfactory, and lens placodes, lens progenitor cells and three-dimensional (3D) primitive lenses, “lentoid bodies”, and “micro-lenses”. These cells are produced alone or “community-grown” with other ocular tissues. Lentoid bodies/micro-lenses generated from human patients carrying mutations in crystallin genes demonstrate proof-of-principle that these cells are suitable for mechanistic studies of cataractogenesis. Taken together, current and emerging advanced in vitro differentiation methods pave the road to understand molecular mechanisms of cataract formation caused by the entire spectrum of mutations in DNA-binding regulatory genes, such as PAX6, SOX2, FOXE3, MAF, PITX3, and HSF4, individual crystallins, and other genes such as BFSP1, BFSP2, EPHA2, GJA3, GJA8, LIM2, MIP, and TDRD7 represented in human cataract patients. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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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 3042

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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19 pages, 1416 KiB

Open AccessEditor’s ChoiceReview

A Linkage between Angiogenesis and Inflammation in Neovascular Age-Related Macular Degeneration

by Hanna Heloterä and Kai Kaarniranta

Cells 2022, 11(21), 3453; https://doi.org/10.3390/cells11213453 - 1 Nov 2022

Cited by 45 | Viewed by 6955

Abstract

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the aging population with a limited understanding of its pathogenesis and the number of patients are all the time increasing. AMD is classified into two main forms: dry and neovascular AMD [...] Read more.

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the aging population with a limited understanding of its pathogenesis and the number of patients are all the time increasing. AMD is classified into two main forms: dry and neovascular AMD (nAMD). Dry AMD is the most prevalent form (80–90%) of AMD cases. Neovascular AMD (10–20% of AMD cases) is treated with monthly or more sparsely given intravitreal anti-vascular endothelial growth factor inhibitors, but unfortunately, not all patients respond to the current treatments. A clinical hallmark of nAMD is choroidal neovascularization. The progression of AMD is initially characterized by atrophic alterations in the retinal pigment epithelium, as well as the formation of lysosomal lipofuscin and extracellular drusen deposits. Cellular damage caused by chronic oxidative stress, protein aggregation and inflammatory processes may lead to advanced geographic atrophy and/or choroidal neovascularization and fibrosis. Currently, it is not fully known why different AMD phenotypes develop. In this review, we connect angiogenesis and inflammatory regulators in the development of nAMD and discuss therapy challenges and hopes. Full article

(This article belongs to the Special Issue The Role of Inflammation in AMD (Multifactorial Age-Related Macular Degeneration))

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