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The immune system exhibits spatial diversity in in vivo tissues. Immune cells are strategically distributed within tissues to maintain the organ integrity. Advanced technologies such as intravital imaging and spatial transcriptomics have revealed the spatial heterogeneity of immune cell distribution and function within organs such as the liver, kidney, intestine, and lung. In addition, these technologies visualize nutrient and oxygen environments across tissues. Recent spatial analyses have suggested that a functional immune niche is determined by interactions between immune and non-immune cells in an appropriate nutrient and oxygen environment. Understanding the spatial communication between immune cells, environment, and surrounding non-immune cells is crucial for developing strategies to control immune responses and effectively manage inflammatory diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Promyelocytic leukemia (PML) protein forms the scaffold for PML nuclear bodies (PML NB) that reorganize into Lipid-Associated PML Structures (LAPS) under fatty acid stress. We determined how the fatty acid oleate alters the interactome of PMLI or PMLII by expressing fusions with the ascorbate peroxidase APEX2 in U2OS cells. The resultant interactome included ESCRT and COPII transport protein nodes. Proximity ligation assay (PLA) revealed that COPII proteins SEC23B, SEC24A and USO1 preferentially associated with PML NBs. Nuclear localization of USO1, but not SEC23B and SEC24A, was reduced in PML knockout cells and restored by PMLII expression. Thus, proximity-labelling methods identified COPII transport protein interactions with PML NBs that are disrupted by fatty acid stress., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Brain metastases (BMs) represent the most prevalent intracranial malignancy within the adult. They are identified in up to 20% of patients with solid tumors and this percentage varies between tumor types and age. Due to the selective permeability of the blood-brain barrier, most anticancer drugs can't reach significant concentrations in the brain, representing a major obstacle to the patients' survival. Furthermore, intra- and inter-patient heterogeneity and the unique brain microenvironment add a layer of complexity to the clinical management of BMs. In the perspective of finding new therapeutic approaches and better understanding the molecular mechanisms involved in brain metastasis, the use of appropriate preclinical models is essential. Here, we review current in vivo, in vitro and ex vivo models for the study of brain metastasis while outlining their advantages and limitations., Competing Interests: Declarations. Conflict of interest: The authors have no conflict of interest to report., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Thyroglobulin is a major autoantigen to which autoimmune response, destroying the thyroid gland in Hashimoto's thyroiditis, is directed. To detect a pathological autoimmune response to thyroglobulin, as well as the successful induction of experimental autoimmune thyroiditis, thyroglobulin carrying thyroiditogenic epitopes is necessary. It is not known which features of thyroglobulin structure determine the presence of thyroiditogenic epitopes and can serve as markers of their presence. We compared structure of thyroglobulin bearing thyroiditogenic epitopes (freshly isolated thyroglobulin) and thyroglobulin which had lost thyroiditogenic epitopes (lyophilized thyroglobulin). Fourier-transform infrared (FTIR) spectroscopy was used to elucidate the structure of thyroglobulin. The markers indicating the presence of thyroiditogenic epitopes on thyroglobulin are the vibrations of diiodotyrosine, monoiodotyrosine/diiodotyrosine relation in the range of 0.24-0.43 (95% confidence interval) and relatively high (> 32%) α-helix content. The loss of thyroiditogenic epitopes on thyroglobulin is associated with a weakening or complete disappearance of diiodotyrosine oscillations and a decrease in the proportion of α-helices in secondary structure. Thyroglobulin extracted with phenylmethylsulfonyl fluoride (PMSF) added is characterized by the same relatively high monoiodotyrosine/diiodotyrosine relation and low proportion of alpha helices as thyroglobulin without thyroiditogenic epitopes. Therefore, serine protease inhibitor PMSF is not suitable for extraction of native thyroglobulin bearing thyroiditogenic epitopes. FTIR spectroscopy can be used to detect thyroiditogenic epitopes on thyroglobulin., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Human milk contains extracellular vesicles (EVs) that carry bioactive molecules such as microRNA, to the newborn intestine. The downstream effects of EV cargo on signaling and immune modulation may shield neonates against inflammatory diseases, including necrotizing enterocolitis. Premature infants are especially at risk, while human milk-feeding may offer protection. The effect of gestational-age specific term and preterm EVs from transitional human milk was characterized on human intestinal epithelial cells (HIECs and Caco-2), primary macrophages, and THP-1 monocytes. We hypothesized that term and preterm EVs differentially influence immune-related cytokines and cell death. We found that preterm EVs were enriched in CD14 surface marker, while both term and preterm EVs increased epidermal growth factor secretion. Following inflammatory stimuli, only term EVs inhibited secretion of IL-6 in HIECs, and reduced expression of pro-inflammatory cytokine IL-1β in macrophages. Term and preterm EVs inhibited secretion of IL-1β and reduced inflammasome related cell death. We proposed that human milk EVs regulate immune-related signaling via their conserved microRNA cargo, which could promote tolerance and a homeostatic immune response. These findings provide basis for further studies into potential therapeutic supplementation with EVs in vulnerable newborn populations by considering functional, gestational age-specific effects. IMPACT: This study reveals distinct functional differences between term and preterm transitional human milk extracellular vesicles (EVs) highlighting the importance of gestational age in their bioactivity. Term EVs uniquely inhibited IL-6 secretion, IL-1β expression, and apoptosis following inflammatory stimuli. Both term and preterm human milk EVs reduced IL-1β secretion and inflammasome-induced cell death. Conserved human milk extracellular vesicle microRNA cargo could be a mediator of the anti-inflammatory effects, particularly targeting cytokine production, the inflammasome, and programmed cell death. These findings underscore the importance of considering gestational age in future research exploring the therapeutic potential of human milk extracellular vesicles to prevent or treat intestinal inflammatory diseases in neonates., Competing Interests: Competing interests: The authors declare no competing interests. Consent for publication: Written informed consent was obtained from all donors prior to their participation in the study., (© 2024. The Author(s).)

Optimization of oncolytic viruses for therapeutic applications requires the strategic removal or mutagenesis of virulence genes alongside the insertion of transgenes that enhance viral replication, spread and immunogenicity. However, the complexity of many viral genomes and the labour-intensive nature of methods for the generation and isolation of recombinant viruses have hindered the development of therapeutic oncolytic viruses. Here we report an iterative strategy that exploits the preferential susceptibility of viruses to certain antibiotics to accelerate the engineering of the genomes of oncolytic viruses for the insertion of immunomodulatory cytokine transgenes, and the identification of dispensable genes with regard to replication of the recombinant oncolytic viruses in tumour cells. We applied the strategy by leveraging insertional mutagenesis via the Sleeping Beauty transposon system, combined with long-read nanopore sequencing, to generate libraries of herpes simplex virus type 1 and vaccinia virus, identifying stable transgene insertion sites and gene deletions that enhance the safety and efficacy of the viruses., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Cell-cell fusion is a vital biological process where the membranes of two or more cells merge to form a syncytium. This phenomenon is critical in various physiological and pathological contexts, including embryonic development, tissue repair, immune responses, and the progression of several diseases. Osteoclasts, which are cells from the monocyte/macrophage lineage responsible for bone resorption, have enhanced functionality due to cell fusion. Additionally, other multinucleated giant cells (MGCs) also arise from the fusion of monocytes and macrophages, typically during chronic inflammation and reactions to foreign materials such as prostheses or medical devices. Foreign body giant cells (FBGCs) and Langhans giant cells (LGCs) emerge only under pathological conditions and are involved in phagocytosis, antigen presentation, and the secretion of inflammatory mediators. This review provides a comprehensive overview of the mechanisms underlying the formation of multinucleated cells, with a particular emphasis on macrophages and osteoclasts. Elucidating the intracellular structures, signaling cascades, and fusion-mediating proteins involved in cell-cell fusion enhances our understanding of this fundamental biological process and helps identify potential therapeutic targets for disorders mediated by cell fusion., Competing Interests: Declarations. Ethics approval and consent to participate: The parabiosis experiment was approved by the Institutional Animal Experimental Committee of Osaka University. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

In most organs, resources such as nutrients, oxygen and physiologically active substances are unevenly supplied within the tissue spaces. Consequently, different tissue functions are exhibited in each space. This spatial heterogeneity of tissue environments arises depending on the spatial arrangement of nutrient vessels and functional vessels, leading to continuous changes in the metabolic states and functions of various cell types from regions proximal to these vessels to distant regions. This phenomenon is referred to as 'zonation'. Traditional analytical methods have made it difficult to investigate this zonation in detail. However, recent advancements in intravital imaging, spatial transcriptomics and single-cell transcriptomics technologies have facilitated the discovery of 'zones' in various organs and elucidated their physiological roles. Here, we outline the spatial differences in the immune system within each zone of organs. This information provides a deeper understanding of organs' immune systems., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The 67 kDa laminin receptor (67LR) was identified as the first laminin receptor shown to be involved in the carcinogenesis of various cancers, including colorectal cancer. While the exact composition of this 67 kDa receptor remains unknown, it has been reported to be formed by the 37 kDa ribosomal protein SA (RPSA) covalently attached to another unidentified protein. The goal of this study was to clarify the molecular structure of 67LR to enhance our understanding of its role in malignancies. Using cell fractionation of colorectal cancer cells, the 67 kDa immunoreactive protein corresponding to 67LR was found in the soluble protein fraction, while some of the 37 kDa RPSA exhibited plasma membrane-like properties. Proteomic analysis of the 67 kDa fraction revealed the absence of RPSA but identified the β-galactosidase-related 67 kDa elastin-binding protein (67EBP), another laminin binding receptor which presents amino acid sequence similarities that can explain the immune cross reactivity with RPSA. The downregulation of β-galactosidase through short hairpin RNA (shRNA) led to a reduction in both 67LR and 67EBP immunoreactive proteins, confirming the misidentification of 67LR and 67EBP in colorectal cancer cells. Based on these findings, we propose to redefine the 67LR as the RPSA-containing laminin receptor (RCLR) to avoid confusion with the 67EBP.

Maintenance of bone homeostasis and the balance between bone resorption and formation are crucial for maintaining skeletal integrity. This study sought to investigate the role of salt-inducible kinase 3 (SIK3), a key regulator in cellular energy metabolism, during the differentiation of osteoclasts. Despite osteoclasts being high energy-consuming cells essential for breaking down mineralized bone tissue, the specific function of SIK3 in this process remains unclear. To address this issue, we generated osteoclast-specific SIK3 conditional knockout mice and assessed the impact of SIK3 deletion on bone homeostasis. Our findings revealed that SIK3 conditional knockout mice exhibited increased bone mass and an osteopetrosis phenotype, suggesting a pivotal role for SIK3 in bone resorption. Moreover, we assessed the impact of pterosin B, a SIK3 inhibitor, on osteoclast differentiation. The treatment with pterosin B inhibited osteoclast differentiation, reduced the numbers of multinucleated osteoclasts, and suppressed resorption activity in vitro. Gene expression analysis demonstrated that SIK3 deletion and pterosin B treatment influence a common set of genes involved in osteoclast differentiation and bone resorption. Furthermore, pterosin B treatment altered intracellular metabolism, particularly affecting key metabolic pathways, such as the tricarboxylic acid cycle and oxidative phosphorylation. These results provide valuable insights into the involvement of SIK3 in osteoclast differentiation and the molecular mechanisms underlying osteoclast function and bone diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background and Objective: Acute myelogenous leukemia (AML) is a common blood cancer marked by heterogeneity in disease and diverse genetic abnormalities. Additional therapies are needed as the 5-year survival remains below 30%. Trametinib is a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor that is widely used in solid tumors and also in tumors with activating RAS mutations. A subset of patients with AML carry activating RAS mutations; however, a small-scale clinical trial with trametinib showed little efficacy. Here, we sought to identify transcriptomic determinants of trametinib sensitivity in AML., Methods: We tested the activity of trametinib against a panel of tumor cells from patients with AML ex vivo and compared this with RNA sequencing (RNA-Seq) data from untreated blasts from the same patient samples. We then used a correlation analysis between gene expression and trametinib sensitivity to identify potential biomarkers predictive of drug response., Results: We found that a subset of AML tumor cells were sensitive to trametinib ex vivo, only a fraction of which (3/10) carried RAS mutations. On the basis of our RNA-Seq analysis we found that markers of trametinib sensitivity are associated with a myeloid differentiation profile that includes high expression of CD14 and CLEC7A (Dectin-1), similar to the gene expression profile of monocytes. Further characterization confirmed that trametinib-sensitive samples display features of monocytic differentiation with high CD14 surface expression and were enriched for the M4 subtypes of the FAB classification., Conclusions: Our study identifies additional molecular markers that can be used with molecular features including RAS status to identify patients with AML that may benefit from trametinib treatment., (© 2024. The Author(s).)

T peripheral helper (Tph) cells are a newly discovered subtype of CD4+ T cells that have emerged as the counterpart of T follicular helper (Tfh) cells in the peripheral tissues. These two cell types share some common characteristics, such as high levels of PD1 and CXCL13 expression, but differ in the expression of transcription factors and chemokine receptors. Tph cells have been studied in relation to B cells' effector functions, including cytokines production and antibody-mediated immune responses. However, their role in the inflammatory-mediated development of malignancies remains poorly understood. Tph cells were initially identified in the synovium of rheumatoid arthritis patients and have since been found to be expanded in several autoimmune diseases. They have been linked to a worse prognosis in autoimmune conditions, but intriguingly, their presence has been correlated with better outcomes in certain types of cancer. The functions of Tph cells are still being investigated, but recent data suggests their involvement in the assembly of tertiary lymphoid structures (TLS). Furthermore, their interaction with B cells, which have been mainly described as possessing a memory phenotype, promotes their development. In this review, we explore the role of Tph cells in peripheral immune responses during cancer and autoimmune disorders., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Background: Individuals affected by the post-covid condition (PCC) show an increased fatigue and the so-called post-exertion malaise (PEM) that led health professionals to advise against exercise although accumulating evidence indicates the contrary. The goal of this study is to determine the impact of a closely monitored 8-week mixed exercise program on physical capacity, symptoms, fatigue, systemic oxidative stress and plasma proteomic profiles of PCC cases., Methods: Twenty-five women and men with PCC were assigned sequentially to exercise ( n = 15) and non-exercise ( n = 10) groups. Individuals with no PCC served as a control group. The exercise program included cardiovascular and resistance exercises. Physical capacity, physical activity level and the presence of common PCC symptoms were measured before and after the intervention. Fatigue was measured the day following each exercise session. Plasma and PBMC samples were collected at the beginning and end of the training program. Glutathione and deoxyguanosine levels in PBMC and plasma proteomic profiles were evaluated., Results: Bicep Curl (+15% vs 4%; p = 0.040) and Sit-to-Stand test (STS-30 (+31% vs +11%; p = 0.043)) showed improvement in the exercise group when compared to the non-exercise group. An interaction effect was also observed for the level of physical activity ( p =0.007) with a positive effect of the program on their daily functioning and without any adverse effects on general or post-effort fatigue. After exercise, glutathione levels in PBMCs increased in women but remained unchanged in men. Discernable changes were observed in the plasma proteomics profile with certain proteins involved in inflammatory response decreasing in the exercise group., Conclusions: Supervised exercise adapted to the level of fatigue and ability is safe and effective in PCC patients in improving their general physical capacity and wellbeing. Systemic molecular markers that accompany physical improvement can be monitored by analyzing plasma proteomics and markers of oxidative stress. Large-scale studies will help identify promising molecular markers to objectively monitor patient improvement., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Chowdhury, Fontaine, Lord, Quenum, Limoges, Rioux-Perreault, Lucier, Cliche, Levesque, Boisvert, Cantin, Allard-Chamard, Menendez, Ilangumaran, Piché, Dionne and Ramanathan.)

Attending and presenting at conferences is a preeminent experience during scientific training. This article provides a trainee's perspective on strategies to promote trainee growth before, during and after scientific meetings, taking the initiatives implemented by the Canadian Society for Immunology (CSI) as an example. A foremost action was the establishment of the Trainee Engagement Committee (TEC) in 2020. The TEC members contribute to the annual symposia by participating in the local organizing committee meetings and organizing trainee-directed events. We propose actions that conference organizers can take to foster the development of the next generation of scientists. In addition, we offer advice to conference presenters on how to craft talks with trainees in mind and to attendees on how to maximize the quality and longevity of conference interactions. We hope this opinion piece evokes reflections and discussions among scientific societies, organizing committees, conference presenters and trainees alike., (© 2024 the Australian and New Zealand Society for Immunology, Inc.)

Background: It has been suggested that the acute natural killer (NK) cell response to aerobic exercise might contribute to the tumor suppressor effect of regular exercise observed in preclinical studies. Moreover, because this response is modulated by exercise intensity, high-intensity intervals exercise (HIIE) might represent an interesting therapeutic approach in cancer patients. However, this immune response remains unstudied in cancer patients currently undergoing chemotherapy., Objective: To characterize the acute NK cell response following a moderate-intensity continuous aerobic exercise session (MOD), and a HIIE session in metastatic cancer patients treated with chemotherapy., Methods: Twelve cancer patients (45-65 years old) underwent a MOD and a duration and work-matched HIIE trial, in a block-randomized order. Peripheral blood mononuclear cells (PBMC) were isolated before, after and 1h after each trial. NK cell subsets were enumerated using flow cytometry and complete blood counts. The surface expression of the cytotoxic NK cell (cNK; CD56 dim CD16 + ) subset was evaluated for its expression of the differentiation markers CD57 and CD158a, the activating receptor NKG2D, the immune checkpoints TIM-3 and PD-1, and the chemokine receptors CXCR3, CXCR4 and CCR2., Results: cNK cell blood counts increased immediately following MOD (p < 0.001) and decreased back to pre-exercise values 1 h after exercise cessation (p < 0.001). The most responsive cNK cell subsets were expressing CD57, CD158a, NKG2D, TIM-3 and CXCR3. The HIIE trial elicited a similar biphasic response, without any difference between trials (all p ≥ 0.38). However, significant changes in the MFI values of CXCR4 and NKG2D were observed in the cNK cell subset following HIIE (all p ≤ 0.038), but not MOD., Conclusion: In metastatic cancer patients undergoing chemotherapy, both MOD and HIIE can elicit an acute mobilisation and egress of NK cells exhibiting phenotypic characteristics associated with high cytotoxicity and tumor homing. Future longitudinal trials are needed to determine if combining aerobic exercise training and chemotherapy will translate towards favorable immune and clinical outcomes., Competing Interests: There are no conflict of interest among all authors., (© 2024 The Authors.)

Lung parenchymal hypoxia has emerged as a cardinal feature of idiopathic pulmonary fibrosis (IPF). Hypoxia promotes cancer cell invasion and metastasis through signaling that is dependent upon the lysophosphatidic acid (LPA) receptor, LPA 1 (LPAR1). Abundant data indicate that LPA 1 -dependent signaling also enhances lung fibrogenesis in IPF. We recently reported that fibroblasts isolated from the lungs of individuals with IPF have an increased capacity to form subcellular matrix-degradative structures known as invadosomes, an event that correlates with the degree of lung fibrosis. We therefore hypothesized that hypoxia promotes invadosome formation in lung fibroblasts through LPA 1 -dependent signaling. Here, it is demonstrated that invadosome formation by fibroblasts from the lungs of individuals with advanced IPF is inhibited by both the tyrosine receptor kinase inhibitor nintedanib and inhibition of LPA 1 . In addition, exposure of normal human lung fibroblasts to either hypoxia or LPA increased their ability to form invadosomes. Mechanistically, the hypoxia-induced invadosome formation by lung fibroblasts was found to involve LPA 1 and PDGFR-Akt signaling. We concluded that hypoxia increases the formation of invadosomes in lung fibroblasts through the LPA 1 and PDGFR-Akt signaling axis, which represents a potential target for suppressing lung fibrosis.

Cumulative research findings support the idea that endocytic trafficking is crucial in regulating receptor signaling and associated diseases. Specifically, strong evidence points to the involvement of sorting nexins (SNXs), particularly SNX1 and SNX2, in the signaling and trafficking of the receptor tyrosine kinase (RTK) MET in colorectal cancer (CRC). Activation of hepatocyte growth factor (HGF) receptor MET is a key driver of CRC progression. In the present study, we utilized human HCT116 CRC cells with SNX1 and SNX2 genes knocked out to demonstrate that their absence leads to a delay in MET entering early endosomes. This delay results in increased phosphorylation of both MET and AKT upon HGF stimulation, while ERK1/2 (extracellular signal-regulated kinases 1 and 2) phosphorylation remains unaffected. Despite these changes, HGF-induced cell proliferation, scattering, and migration remain similar between the parental and the SNX1/2 knockout cells. However, in the absence of SNX1 and SNX2, these cells exhibit increased resistance to TRAIL-induced apoptosis. This research underscores the intricate relationship between intracellular trafficking, receptor signaling, and cellular responses and demonstrates for the first time that the modulation of MET trafficking by SNX1 and SNX2 is critical for receptor signaling that may exacerbate the disease., (© 2024 The Author(s).)

The function of germ cells in somatic growth and aging has been demonstrated in invertebrate models but remains unclear in vertebrates. We demonstrated sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri . In females, germ cell removal shortened life span, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ cell removal in males improved their health with increased vitamin D signaling. Body size increased in both sexes but was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different pathways of the endocrine system, depending on the sex, which may underlie the sexual difference in reproductive strategies.

Background: Inflammatory cytokines play key pathogenic roles in liver fibrosis. IL-15 is a proinflammatory cytokine produced by myeloid cells. IL-15 promotes pathogenesis of several chronic inflammatory diseases. However, increased liver fibrosis has been reported in mice lacking IL-15 receptor alpha chain (IL-15Rα), suggesting an anti-fibrogenic role for IL-15. As myeloid cells are key players in liver fibrosis and IL-15 signaling can occur independently of IL-15Rα, we investigated the requirement of IL-15 and IL-15Rα in liver fibrosis., Methods: We induced liver fibrosis in Il15 -/- , Il15ra -/- and wildtype C57BL/6 mice by the administration of carbon tetrachloride (CCl 4 ). Liver fibrosis was evaluated by Sirius red and Mason's trichrome staining and α-smooth muscle acting immunostaining of myofibroblasts. Gene expression of collagens, matrix modifying enzymes, cytokines and chemokines was quantified by RT-qPCR. The phenotype and the numbers of intrahepatic lymphoid and myeloid cell subsets were evaluated by flow cytometry., Results: Both Il15 -/- and Il15ra -/- mice developed markedly reduced liver fibrosis compared to wildtype control mice, as revealed by reduced collagen deposition and myofibroblast content. Il15ra -/- mice showed further reduction in collagen deposition compared to Il15 -/- mice. However, Col1a1 and Col1a3 genes were similarly induced in the fibrotic livers of wildtype, Il15 -/- and Il15ra -/- mice, although notable variations were observed in the expression of matrix remodeling enzymes and chemokines. As expected, Il15 -/- and Il15ra -/- mice showed markedly reduced numbers of NK cells compared to wildtype mice. They also showed markedly less staining of CD45 + immune cells and CD68 + macrophages, and significantly reduced inflammatory cell infiltration into the liver, with fewer pro-inflammatory and anti-inflammatory monocyte subsets compared to wildtype mice., Conclusion: Our findings indicate that IL-15 exerts its profibrogenic role in the liver by promoting macrophage activation and that this requires trans-presentation of IL-15 by IL-15Rα., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cloutier, Variya, Akbari, Rexhepi, Ilangumaran and Ramanathan.)

Pancreaticobiliary cancer, encompassing malignancies of both the pancreatic and biliary tract, presents a formidable clinical challenge marked by a uniformly bleak prognosis. The asymptomatic nature of its early stages often leads to delayed detection, contributing to an unfavorable 5-year overall survival rate. Conventional treatment modalities have shown limited efficacy, underscoring the urgent need for alternative therapeutic approaches. In recent years, immunotherapy has emerged as a promising avenue in the fight against pancreaticobiliary cancer. Strategies such as therapeutic vaccines and the use of tumor-infiltrating lymphocytes have garnered attention for their potential to elicit more robust and durable responses. This review seeks to illuminate the landscape of emerging immunotherapeutic interventions, offering insights from both clinical and research perspectives. By deepening our understanding of pancreaticobiliary cancer and exploring innovative treatment modalities, we aim to catalyze improvements in patient outcomes and quality of life., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

To identify credible causal risk variants (CCVs) associated with different histotypes of epithelial ovarian cancer (EOC), we performed genome-wide association analysis for 470,825 genotyped and 10,163,797 imputed SNPs in 25,981 EOC cases and 105,724 controls of European origin. We identified five histotype-specific EOC risk regions (p value <5 × 10 -8 ) and confirmed previously reported associations for 27 risk regions. Conditional analyses identified an additional 11 signals independent of the primary signal at six risk regions (p value <10 -5 ). Fine mapping identified 4,008 CCVs in these regions, of which 1,452 CCVs were located in ovarian cancer-related chromatin marks with significant enrichment in active enhancers, active promoters, and active regions for CCVs from each EOC histotype. Transcriptome-wide association and colocalization analyses across histotypes using tissue-specific and cross-tissue datasets identified 86 candidate susceptibility genes in known EOC risk regions and 32 genes in 23 additional genomic regions that may represent novel EOC risk loci (false discovery rate <0.05). Finally, by integrating genome-wide HiChIP interactome analysis with transcriptome-wide association study (TWAS), variant effect predictor, transcription factor ChIP-seq, and motifbreakR data, we identified candidate gene-CCV interactions at each locus. This included risk loci where TWAS identified one or more candidate susceptibility genes (e.g., HOXD-AS2, HOXD8, and HOXD3 at 2q31) and other loci where no candidate gene was identified (e.g., MYC and PVT1 at 8q24) by TWAS. In summary, this study describes a functional framework and provides a greater understanding of the biological significance of risk alleles and candidate gene targets at EOC susceptibility loci identified by a genome-wide association study., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Cancer tissues contain a wide variety of immune cells that play critical roles in suppressing or promoting tumor progression. Macrophages are one of the most predominant populations in the tumor microenvironment and are composed of two classes: infiltrating macrophages from the bone marrow and tissue-resident macrophages (TRMs). This review aimed to outline the function of TRMs in the tumor microenvironment, focusing on lung cancer., Review: Although the functions of infiltrating macrophages and tumor-associated macrophages have been intensively analyzed, a comprehensive understanding of TRM function in cancer is relatively insufficient because it differs depending on the tissue and organ. Alveolar macrophages (AMs), one of the most important TRMs in the lungs, are replenished in situ, independent of hematopoietic stem cells in the bone marrow, and are abundant in lung cancer tissue. Recently, we reported that AMs support cancer cell proliferation and contribute to unfavorable outcomes., Conclusion: In this review, we introduce the functions of AMs in lung cancer and their underlying molecular mechanisms. A thorough understanding of the functions of AMs in lung cancer will lead to improved treatment outcomes., (© 2024. The Author(s).)

Targeting tumor-associated macrophages (TAMs) is an emerging approach being tested in multiple clinical trials. TAMs, depending on their differentiation state, can exhibit pro- or antitumorigenic functions. For example, the M2-like phenotype represents a protumoral state that can stimulate tumor growth, angiogenesis, metastasis, therapy resistance, and immune evasion by expressing immune checkpoint proteins. In this issue of the JCI, Vaccaro and colleagues utilized an innovative drug screen approach to demonstrate that targeting driver oncogenic signaling pathways concurrently with anti-CD47 sensitizes tumor cells, causing them to undergo macrophage-induced phagocytosis. The combination treatment altered expression of molecules on the tumor cells that typically limit phagocytosis. It also reprogrammed macrophages to an M1-like antitumor state. Moreover, the approach was generalizable to tumor cells with different oncogenic pathways, opening the door to precision oncology-based rationale combination therapies that have the potential to improve outcomes for patients with oncogene-driven lung cancers and likely other cancer types.

The liver is the main gateway from the gut, and the unidirectional sinusoidal flow from portal to central veins constitutes heterogenous zones, including the periportal vein (PV) and the pericentral vein zones 1-5 . However, functional differences in the immune system in each zone remain poorly understood. Here intravital imaging revealed that inflammatory responses are suppressed in PV zones. Zone-specific single-cell transcriptomics detected a subset of immunosuppressive macrophages enriched in PV zones that express high levels of interleukin-10 and Marco, a scavenger receptor that sequesters pro-inflammatory pathogen-associated molecular patterns and damage-associated molecular patterns, and consequently suppress immune responses. Induction of Marco + immunosuppressive macrophages depended on gut microbiota. In particular, a specific bacterial family, Odoribacteraceae, was identified to induce this macrophage subset through its postbiotic isoallolithocholic acid. Intestinal barrier leakage resulted in inflammation in PV zones, which was markedly augmented in Marco-deficient conditions. Chronic liver inflammatory diseases such as primary sclerosing cholangitis (PSC) and non-alcoholic steatohepatitis (NASH) showed decreased numbers of Marco + macrophages. Functional ablation of Marco + macrophages led to PSC-like inflammatory phenotypes related to colitis and exacerbated steatosis in NASH in animal experimental models. Collectively, commensal bacteria induce Marco + immunosuppressive macrophages, which consequently limit excessive inflammation at the gateway of the liver. Failure of this self-limiting system promotes hepatic inflammatory disorders such as PSC and NASH., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication., Competing Interests: Conflict of interest The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Purpose: Hashimoto's thyroiditis (HT) is an autoimmune thyroid disease characterized by T lymphocyte-mediated destruction of thyroid follicles. To study the pathogenesis of HT and the efficacy of new substances for its treatment, an easily obtained and adequate to the human disease experimental model is needed. The aim of our study was to find out whether it is possible to induce experimental autoimmune thyroiditis (EAT) similar to Hashimoto's thyroiditis by injecting with thyroglobulin (Tg) without using agents that enhance its thyroiditogenicity and without taking into account the genetic sensitivity of animals., Methods: Wistar rats were immunized with freshly isolated rat Tg or porcine Tg. In 8 weeks, histological studies of the thyroid and parathyroid glands were performed. Thyroid function and total serum calcium level were also evaluated., Results: Immunization with both rat and porcine freshly isolated Tg caused T lymphocytic infiltration of the thyroid gland, thyroid follicle atrophy and degradation in Wistar rats. EAT caused by porcine Tg was characterized by greater severity than EAT induced with rat Tg. In 55% of rats with porcine Tg-induced EAT, oxyphilic metaplasia was detected in the parathyroid glands. In addition, low total serum calcium was observed in these rats., Conclusion: Two rat models of autoimmune thyroiditis were obtained. EAT caused in Wistar rats by immunization with rat Tg is similar to Hashimoto's thyroiditis. EAT induced with porcine Tg was accompanied by oxyphil cell metaplasia in the parathyroids and hypocalcemia., (© 2023. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Esophageal squamous cell carcinoma (ESCC) is a deadly consequence of radiation exposure to the esophagus. ESCC arises from esophageal epithelial cells that undergo malignant transformation and features a perturbed squamous cell differentiation program. Understanding the dose- and radiation quality-dependence of the esophageal epithelium response to radiation may provide insights into the ability of radiation to promote ESCC. We have explored factors that may play a role in esophageal epithelial radiosensitivity and their potential relationship to ESCC risk. We have utilized a murine three-dimensional (3D) organoid model that recapitulates the morphology and functions of the stratified squamous epithelium of the esophagus to study persistent dose- and radiation quality-dependent changes. Interestingly, although high-linear energy transfer (LET) Fe ion exposure induced a more intense and persistent alteration of squamous differentiation and 53BP1 DNA damage foci levels as compared to Cs, the MAPK/SAPK stress pathway signaling showed similar altered levels for most phospho-proteins with both radiation qualities. In addition, the lower dose of high-LET exposure also revealed nearly the same degree of morphological changes, even though only ~36% of the cells were predicted to be hit at the lower 0.1 Gy dose, suggesting that a bystander effect may be induced. Although p38 and ERK/MAPK revealed the highest levels following high-LET exposure, the findings reveal that even a low dose (0.1 Gy) of both radiation qualities can elicit a persistent stress signaling response that may critically impact the differentiation gradient of the esophageal epithelium, providing novel insights into the pathogenesis of radiation-induced esophageal injury and early stage esophageal carcinogenesis., Competing Interests: Deepa M. Sridharan is from Soley Theraperutics, Inc. The authors declare no conflicts of interest.

The aim of this study was to analyze the association between vaginal microbiota, carbonic anhydrase IX (CAIX) and histological findings of cervical intraepithelial neoplasia (CIN). The study included 132 females, among them 66 were diagnosed with high-grade intraepithelial lesion (CIN2, CIN3, and cancer), 14 with low-grade disease, and 52 assigned to the control group. An interview focused on the behavior risk factors, together with vaginal fluid pH measurement, wet mount microscopy, detection of Chlamydia trachomatis, and Trichomonas vaginalis were performed. After colposcopy, high-grade abnormalities were detected via direct biopsies and treated with conization procedure. Conuses were immuno-stained with CAIX antibody. The histological findings were CIN1 (n = 14), and CIN2+ (included CIN2 (n = 10), CIN3 (n = 49), and cancer (n = 7; squamous cell carcinomas)). Prevalence of bacterial vaginosis (BV) was similar between the groups. Moderate or severe aerobic vaginitis (msAV) was diagnosed more often among CIN2+ (53.0%) than CIN1 (21.4%). Moderate or strong immunostaining of CAIX (msCAIX) was not detected among CIN1 cases. Thus, msAV was prevalent in CAIX non-stained group (p = 0.049) among CIN2 patients. Co-location of msAV and msCAIX was found in CIN3. Regression model revealed that msAV associated with high-grade cervical intraepithelial neoplasia independently from smoking and the number of partners., (© 2024. The Author(s).)

Understanding the complex network of protein-protein interactions (PPI) that govern cellular functions is essential for unraveling the molecular basis of biological processes and diseases. Mass spectrometry (MS) has emerged as a powerful tool for studying protein dynamics, enabling comprehensive analysis of protein function, structure, post-translational modifications, interactions, and localization. This article provides an overview of MS techniques and their applications in proteomics studies, with a focus on the replication fork proteome. The replication fork is a multi-protein assembly involved in DNA replication, and its proper functioning is crucial for maintaining genomic integrity. By combining quantitative MS labeling techniques with various data acquisition methods, researchers have made significant strides in elucidating the complex processes and molecular mechanisms at the replication fork. Overall, MS has revolutionized our understanding of protein dynamics, offering valuable insights into cellular processes and potential targets for therapeutic interventions., Competing Interests: The authors declare no competing interests.

Recently, we reported that during the hypertrophic phase (230 days old) of hereditary cardiomyopathy of the hamster (HCMH), short-term treatment (20 days) with 250 mg/kg/day of taurine prevents the development of hypertrophy in males but not in females. However, the mortality rate in non-treated animals was higher in females than in males. To verify whether the sex-dependency effect of taurine is due to the difference in the disease's progression, we treated the 230-day-old animals for a longer time period of 122 days. Our results showed that long-term treatment with low and high concentrations of taurine significantly prevents cardiac hypertrophy and early death in HCMH males ( p < 0.0001 and p < 0.05, respectively) and females ( p < 0.01 and p < 0.0001, respectively). Our results demonstrate that the reported sex dependency of short-term treatments with taurine is due to a higher degree of heart remodeling in females when compared to males and not to sex dependency. In addition, sex-dependency studies should consider the differences between the male and female progression of the disease. Thus, long-term taurine therapies are recommended to prevent remodeling and early death in hereditary cardiomyopathy.

Endocardial endothelium (EE) is a layer of cells covering the cardiac cavities and modulates cardiomyocyte function. This cell type releases several cardioactive factors, including Angiotensin II (Ang II). This octopeptide is known to induce cardiac hypertrophy. However, whether this circulating factor also induces EE hypertrophy is not known. Taurine is known to prevent cardiac hypertrophy. Whether this endogenous antioxidant prevents the effect of Ang II on human EE (hEE) will be verified. Using quantitative fluorescent probe imaging for calcium and reactive oxygen species (ROS), our results show that Ang II induces (10 -7 M, 48 h treatment) an increase in hEE cell (hEEC) volume and its nucleus. Pretreatment with 20 mM of taurine prevents morphological remodeling and increases intracellular calcium and ROS. These results suggest that the reported Ang II induces cardiac hypertrophy is associated with hEEC hypertrophy. This later effect is prevented by taurine by reducing intracellular calcium and ROS overloads. Thus, taurine could be an excellent tool for preventing Ang II-induced remodeling of hEECs.

Suppressor of cytokine signaling 1 (SOCS1) is a potent regulator immune cell responses and a proven tumor suppressor. Inhibition of SOCS1 in T cells can boost antitumor immunity, whereas its loss in tumor cells increases tumor aggressivity. Investigations into the tumor suppression mechanisms so far focused on tumor cell-intrinsic functions of SOCS1. However, it is possible that SOCS1 expression in tumor cells also regulate antitumor immune responses in a cell-extrinsic manner via direct and indirect mechanisms. Here, we discuss the evidence supporting the latter, and its implications for antitumor immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ilangumaran, Gui, Shukla and Ramanathan.)

Inflammatory bowel disease (IBD) flare-ups exhibit symptoms that are similar to other diseases and conditions, making diagnosis and treatment complicated. Currently, the gold standard for diagnosing and monitoring IBD is colonoscopy and biopsy, which are invasive and uncomfortable procedures, and the fecal calprotectin test, which is not sufficiently accurate. Therefore, it is necessary to develop an alternative method. In this study, our aim was to provide proof of concept for the application of Sequential Window Acquisition of All Theoretical Mass Spectra-Mass spectrometry (SWATH-MS) and machine learning to develop a non-invasive and accurate predictive model using the stool proteome to distinguish between active IBD patients and symptomatic non-IBD patients. Proteome profiles of 123 samples were obtained and data processing procedures were optimized to select an appropriate pipeline. The differentially abundant analysis identified 48 proteins. Utilizing correlation-based feature selection (Cfs), 7 proteins were selected for proceeding steps. To identify the most appropriate predictive machine learning model, five of the most popular methods, including support vector machines (SVMs), random forests, logistic regression, naive Bayes, and k-nearest neighbors (KNN), were assessed. The generated model was validated by implementing the algorithm on 45 prospective unseen datasets; the results showed a sensitivity of 96% and a specificity of 76%, indicating its performance. In conclusion, this study illustrates the effectiveness of utilizing the stool proteome obtained through SWATH-MS in accurately diagnosing active IBD via a machine learning model.

The esophagus is protected from the hostile environment by a stratified epithelium, which renews rapidly. Homeostasis of this epithelium is ensured by a rare population of stem cells in the basal layer: Keratin 15+ (Krt15+) cells. However, little is known about the molecular mechanisms regulating their distinct features, namely self-renewal, potency and epithelial regeneration. Achaete-scute family BHLH transcription factor 2 (ASCL2) is strongly upregulated in Krt15+ stem cells and is known to contribute to stem cell maintenance in other tissues. Herein, we investigated the role of ASCL2 in maintaining homeostasis under normal and stress conditions in the esophageal epithelium. ASCL2 overexpression severely dysregulated cell differentiation and cell fate. Proliferation was also reduced due potentially to a blockage in the G1 phase of the cell cycle or an induction of quiescence. Mass spectrometry analysis confirmed alterations in several proteins associated with differentiation and the cell cycle. In addition, overexpression of ASCL2 enhanced resistance to radiation and chemotherapeutic drugs. Overall, these results denote the role of ASCL2 as a key regulator of the proliferation-differentiation equilibrium in the esophageal epithelium., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

SOCS1 is a tumor suppressor in hepatocellular carcinoma (HCC). Recently, we showed that a loss of SOCS1 in hepatocytes promotes NRF2 activation. Here, we investigated how SOCS1 expression in HCC cells affected oxidative stress response and modulated the cellular proteome. Murine Hepa1-6 cells expressing SOCS1 (Hepa-SOCS1) or control vector (Hepa-Vector) were treated with cisplatin or tert-butyl hydroperoxide ( t -BHP). The induction of NRF2 and its target genes, oxidative stress, lipid peroxidation, cell survival and cellular proteome profiles were evaluated. NRF2 induction was significantly reduced in Hepa-SOCS1 cells. The gene and protein expression of NRF2 targets were differentially induced in Hepa-Vector cells but markedly suppressed in Hepa-SOCS1 cells. Hepa-SOCS1 cells displayed an increased induction of reactive oxygen species but reduced lipid peroxidation. Nonetheless, Hepa-SOCS1 cells treated with cisplatin or t -BHP showed reduced survival. GCLC , poorly induced in Hepa-SOCS1 cells, showed a strong positive correlation with NFE2L2 and an inverse correlation with SOCS1 in the TCGA-LIHC transcriptomic data. A proteomic analysis of Hepa-Vector and Hepa-SOCS1 cells revealed that SOCS1 differentially modulated many proteins involved in diverse molecular pathways, including mitochondrial ROS generation and ROS detoxification, through peroxiredoxin and thioredoxin systems. Our findings indicate that maintaining sensitivity to oxidative stress is an important tumor suppression mechanism of SOCS1 in HCC.

Background: Hepatic stellate cells (HSC) are the key mediators of fibrosis development in non-alcoholic fatty liver disease (NAFLD). Hepatic inflammation induced by high-fat diet activates HSCs, which differentiate to myofibroblasts and produce extracellular fibrillar matrix. HSC activation during hepatic fibrogenesis is modulated by cytokines and growth factors produced by stressed hepatocytes and macrophages. SOCS1 is a negative feedback regulator of certain cytokines and growth factors implicated in liver fibrosis., Aim: The goal of this study was to understand the regulatory functions of SOCS1 in HSCs during NAFLD-induced liver fibrosis., Methodology: Mice lacking SOCS1 specifically in HSCs ( Socs1 ) and control ΔHSC ) and control Socs1 ) mice were fed choline-deficient L-amino acid-defined high-fat diet (CDA-HFD) or normal control diet for 14 weeks. Body weight gain was regularly monitored. Serum alanine aminotransferase levels and liver weight were assessed at the endpoint. Fibrosis development was evaluated by Sirius red staining and hydroxyproline content, and myofibroblast differentiation by immunohistochemistry. Expression of genes encoding pro-fibrogenic factors, cytokines, growth factors and chemokines, and the phenotype and numbers of intrahepatic leukocytes were evaluated.Socs1 fl/fl ) mice were fed choline-deficient L-amino acid-defined high-fat diet (CDA-HFD) or normal control diet for 14 weeks. Body weight gain was regularly monitored. Serum alanine aminotransferase levels and liver weight were assessed at the endpoint. Fibrosis development was evaluated by Sirius red staining and hydroxyproline content, and myofibroblast differentiation by immunohistochemistry. Expression of genes encoding pro-fibrogenic factors, cytokines, growth factors and chemokines, and the phenotype and numbers of intrahepatic leukocytes were evaluated., Results: Socs1 mice showed increased liver/body weight ratio and displayed increased collagen deposition and myofibroblast differentiation. Induction of ΔHSC mice showed increased liver/body weight ratio and displayed increased collagen deposition and myofibroblast differentiation. Induction of Acta2 , Col1a1 , Pdgfb and IL1b and Ccl2 genes was significantly elevated in Socs1 ΔHSC mice compared to Socs1 livers displayed increased SMAD3 phosphorylation. The fibrotic livers of fl/fl mice showed increased inflammatory cell infiltration, and flow cytometry analysis revealed elevated numbers of myeloid cells, granulocytes and myeloid-derived dendritic cells. Tgfb livers harbored increased numbers of Ly6C Socs1 CCR2+ pro-inflammatory macrophages, largely comprised of Ly6C ΔHSC livers displayed increased SMAD3 phosphorylation. The fibrotic livers of Socs1 ΔHSC mice showed increased inflammatory cell infiltration, and flow cytometry analysis revealed elevated numbers of myeloid cells, granulocytes and myeloid-derived dendritic cells. Socs1 ΔHSC livers harbored increased numbers of Ly6C hi CCR2+ pro-inflammatory macrophages, largely comprised of Ly6C hi CCR2+CX3CR1+ cells, suggesting impaired transition to anti-inflammatory macrophages., Conclusion: Our findings show that SOCS1 exerts non-redundant regulatory functions in HSCs that are critical for attenuating high-fat diet-induced inflammatory response and liver fibrosis development., (© 2023 Indian National Association for Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Human milk extracellular vesicles (HM EVs) are proposed to protect against disease development in infants. This protection could in part be facilitated by the bioactive EV cargo of proteins and RNA. Notably, mothers birth infants of different gestational ages with unique needs, wherein the EV cargo of HM may diverge. We collected HM from lactating mothers within two weeks of a term or preterm birth. Following purification of EVs, proteins and mRNA were extracted for proteomics and sequencing analyses, respectively. Over 2000 protein groups were identified, and over 8000 genes were quantified. The total number of proteins and mRNA did not differ significantly between the two conditions, while functional bioinformatics of differentially expressed cargo indicated enrichment in immunoregulatory cargo for preterm HM EVs. In term HM EVs, significantly upregulated cargo was enriched in metabolism-related functions. Based on gene expression signatures from HM-contained single cell sequencing data, we proposed that a larger portion of preterm HM EVs are secreted by immune cells, whereas term HM EVs contain more signatures of lactocyte epithelial cells. Proposed differences in EV cargo could indicate variation in mother's milk based on infants' gestational age and provide basis for further functional characterisation., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

The in situ behavior of living cells can be visualized by two-photon microscopy. Here, we present a protocol for the live imaging of transferred mouse bone marrow cells by two-photon microscopy. We describe steps for staining and injecting target cells into mice, fixing the skull bone to a head holder and stage, and 4D imaging bone marrow using multi-photon microscopy. We then detail procedures for creating images and analyzing cells. For complete details on the use and execution of this protocol, please refer to Sudo et al. (2021). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)