Search Results (89)

Background: Phosphaturic mesenchymal tumours secreting fibroblast growth factor 23 (hereinafter referred to as FGF23+ PMT) are rare neoplasms that can cause hypophosphataemic osteomalacia, owing to excessive FGF23 production. Mast cells (MCs) play a key role in tumour biology by modulating proliferative activity of atypical cells, resistance to innate and acquired immunity, angiogenesis, and metastatic behaviour. However, MCs associated with FGF23+ PMT have not previously been investigated. This study, to our knowledge, is the first to characterise features of the tumour microenvironment through spatial phenotyping of the immune and stromal landscape, together with histotopographic mapping of intercellular MC interactions with other subcellular populations in FGF23+ PMT. Methods: Histochemical staining (haematoxylin and eosin, toluidine blue, Giemsa solution, picro-Mallory protocol, silver impregnation), as well as monoplex and multiplex immunohistochemical staining with spatial phenotyping, were performed to detect atypical FGF23-secreting cells, immune cells (CD3, CD4, CD8, CD14, CD20, CD38, CD68, or CD163), stromal components (CD31, α-SMA, or vimentin), and specific MC proteases (tryptase, chymase, or carboxypeptidase A3). Bioinformatics analysis using artificial intelligence technologies was applied for spatial profiling of MC interactions with tumour, immunocompetent, and stromal cells in the tumour microenvironment. Results: Bioinformatic analysis of the entire tumour histological section, comprising over 70,000 cells stained using monoplex and multiplex immunohistochemical protocols, enabled identification of more than half of the cell population. The most abundant were CD14+ (30.7%), CD163+ (23.2%), and CD31+ (17.9%) cells. Tumour-associated MCs accounted for 0.7% of the total pool of immunopositive cells and included both mucosal and connective tissue subpopulations, predominantly of the tryptase + chymase-CPA3-specific protease phenotype. This pattern reflected combined multidirectional morphogenetic processes in the patient’s FGF23+ PMT. More than 50% of MCs were colocalized with neighbouring cells of the tumour microenvironment within 20 μm, most frequently with monocytes (CD14+CD68+), M2 macrophages (CD68+CD163+), and endothelial cells (CD31+). In contrast, colocalization with atypical FGF23-secreting cells was rare, indicating minimal direct effects on tumour cell activity. Interaction with T lymphocytes, including CD8+, was also infrequent, excluding their activation and the development of antitumour effects. Mapping of MC histotopography validated the hypothesis of their inductive role in monocyte differentiation into M2 macrophages and probable polarisation of macrophages from M1 into M2, thereby contributing to slow tumour growth. MCs were further involved in extracellular matrix remodelling and participated in the formation of pro-osteogenic niches within the FGF23+ PMT microenvironment, leading to pathological osteoid development. Conclusions: This study demonstrated active MC participation in the evolution of the FGF23+ PMT microenvironment. The findings may be applied in translational medicine to develop novel algorithms for personalised therapy in patients with FGF23-secreting tumours, offering an alternative when surgical removal of the tumour is not feasible. Full article

Autologous fat grafting of human lipoaspirate (LA) is increasingly used in reconstructive and cosmetic surgery for lipofilling and stem cell-rich “nanofat” reinjection for regenerative medicine. While commercial devices (e.g., REVOLVE and Puregraft) are available, many surgeons use non-standardized manual washing techniques, leading to inconsistent graft retention (20–80%). Moreover, no system can unite washing directly with mechanical processing to produce a nanofat-like product directly from raw LA. We developed a novel preparation device (PD) that is designed for peristaltic pump-driven washing of LA and can be seamlessly combined with our previously developed Emulsification and Micronization Device (EMD) into an automated closed-loop platform. Human LA samples were washed with the PD and compared to standard manual washing via visual colorimetric analysis. We then evaluated the mechanical processing of PD-washed LA using our EMD and assessed cell count, viability, and stromal vascular fraction-derived subpopulations (i.e., mesenchymal stem cells, endothelial progenitor cells (EPCs), pericytes, transit-amplifying (TA) progenitor cells, and supra-adventitial adipose stromal cells). Recirculating LA through the PD for at least one minute resulted in sufficient mixing, producing LA with equivalent color and quality to manual washing. Integrating the EMD within a platform enabled both washing and mechanical processing under peristaltic flow, enriching key subpopulations compared to manual methods. Thus, our fluidic platform effectively washes LA in a closed-loop system, minimizing LA tissue manipulation and opportunity for contamination while also simplifying the workflow for mechanical processing. Further refinement and automation of this platform would enhance the reproducibility and quality of small-volume fat grafts, cell-assisted lipotransfer, and stem/progenitor cell injections to promote wound healing and angiogenesis. Full article

Cutaneous melanoma is an aggressive cancer with an increasing incidence worldwide, highlighting the need for research into its pathogenesis. The tumor microenvironment (TME) plays a critical role in melanoma progression and consists of cellular components and an extracellular matrix (ECM) rich in cytokines and signaling molecules. The most abundant stromal cells within the TME are cancer-associated fibroblasts (CAFs), which remodel the ECM and modulate immune responses. Among immune cells, tumor-associated macrophages (TAMs) predominate, and their polarization toward the M2 phenotype supports tumor progression. Tumor-infiltrating lymphocytes (TILs) have diverse functions, including cytotoxic T-cells, helper T-cells that modulate immune response, B-cells forming tertiary lymphoid structures (TLS), and regulatory T-cells with immunosuppressive properties. Dendritic cells (DCs) also play a complex role in the TME. A notable subpopulation are mature regulatory dendritic cells (mregDCs), which contribute to immune evasion. All of these TME components may drive tumorigenesis. Advancements in melanoma treatment—including immunotherapy and targeted therapies—have significantly improved outcomes in advanced-stage disease. In parallel, emerging approaches targeting the tumor microenvironment and gut microbiome, as well as personalized strategies such as neoantigen vaccines and cell-based therapies, are under active investigation and may further enhance therapeutic efficacy in the near future. Full article

Background/Purpose: Conventional three-dimensional in vitro tumor models often fail to fully capture the complexity of the tumor microenvironment, particularly the diverse populations of cancer-associated fibroblasts that contribute to poor prognosis and treatment resistance. The purpose of this study is to develop a patient-specific gastric cancer assembloid model that integrates tumor epithelial cells with matched stromal cell subtypes, each derived using tailored growth media to enhance cancer preclinical research and advance personalized therapeutic strategies. Methods: Tumor tissue was dissociated, and cells expanded in media for organoids, mesenchymal stem cells, fibroblasts, or endothelial cells. The resulting tumor-derived subpopulations were co-cultured in an optimized assembloid medium supporting each cell type’s growth. Biomarker expression was assessed by immunofluorescence staining, and transcriptomic profiles were analyzed by RNA sequencing. Drug responsiveness was evaluated using cell viability assays following treatment with various therapeutic agents. Results: The optimized co-culture conditions yielded assembloids that closely mimicked the cellular heterogeneity of primary tumors, confirmed by the expression of epithelial and stromal markers. Compared to monocultures, the assembloids showed higher expression of inflammatory cytokines, extracellular matrix remodeling factors, and tumor progression-related genes across different organoids and stromal ratios. Drug screening revealed patient- and drug-specific variability. While some drugs were effective in both organoid and assembloid models, others lost efficacy in the assembloids, highlighting the critical role of stromal components in modulating drug responses. Conclusions: This assembloid system offers a robust platform to study tumor–stroma interactions, identify resistance mechanisms, and accelerate drug discovery and personalized therapeutic strategies for gastric cancer. Full article

Recent advancements in spatial transcriptomics (ST) have revolutionized our understanding of the lung’s cellular organization and pathological alterations. By preserving the spatial distribution of gene expression, ST reveals localized immune niches, stromal–epithelial interactions, and disease-associated transcriptional “hotspots” that cannot be captured by conventional sequencing methods alone. In lung cancer, ST-based investigations have delineated distinct tumor microenvironments between tumor cores and invasive fronts, revealing prognostically significant gene signatures and identifying subpopulations with differential responses to immunotherapy and chemotherapy. Similarly, in chronic obstructive pulmonary disease, asthma, and idiopathic pulmonary fibrosis, ST has mapped the ecosystem, including immune cells, inflammatory mediators, and fibroblast subtypes, of discrete regions within diseased lung tissue, offering mechanistic insights into disease progression and tissue remodeling. In addition, a more recent ST study provides spatial information for where drugs act within tissues. This review highlights the emerging role of spatial transcriptomics in respiratory research, demonstrating its potential to refine disease classification, elucidate mechanisms of therapeutic resistance, and inform spatially guided personalized interventions in respiratory diseases. Full article

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis, and its progression is driven by epithelial plasticity and tumor microenvironment remodeling. Finding biomarkers that are responsible for the turning point from the early stage to the aggressive phase would facilitate clinical management. Method: In this study, we employed single-cell RNA sequencing to characterize a distinct subpopulation of highly proliferative epithelial cells undergoing a transitional phase during PDAC progression. By linking to cell cycle dysregulation, epithelial differentiation, and clinical staging, we constructed a gene-based risk score model using Lasso Cox regression. The expression of selected genes within the model was further validated using qPCR. Results: The model demonstrated robust predictive power for patient prognosis, TNM staging, and chemotherapy sensitivity. Further analysis of the tumor microenvironment revealed intensified crosstalk between a specific fibroblast subpopulation and transitional epithelial cells, mediated largely by collagen signaling. This stromal–epithelial interaction was found to contribute to the fibrotic barrier characteristic of PDAC. Additionally, immune profiling uncovered altered infiltration patterns, particularly involving natural killer (NK) cells in high-risk patients, suggesting mechanisms of immune tolerance and inhibition. Conclusions: These findings offer potential avenues for early detection, risk stratification, and targeted therapeutic strategies in PDAC. Full article

Papillary thyroid cancer (PTC) contains mesenchymal stem/stromal cells (MSCs), but their contribution to PTC progression is not clear. In this study, we compared the transcriptional signatures of normal thyroid (NT) and PTC-derived MSCs with the aim of determining if these have distinct transcriptomes that might influence PTC progression. We used flow cytometry in combination with a panel of MSC clusters of differentiation (CD) markers and showed that both thyroid MSC populations expressed MSC markers and lacked expression of markers not normally expressed by MSCs. In addition, we determined that both MSC populations could differentiate to adipocytes and osteocytes. Analysis of single cell RNA sequencing data from both MSC populations revealed, regardless of tissue of origin, that both contained similar numbers of subpopulations. Cluster analysis revealed similarity in expression of both MSC populations for stromal markers, the vascular marker VEGFA and the smooth muscle marker CALD1, while smaller subpopulations expressed markers of more lineage-committed thyroid cells. PTC MSCs also showed upregulated expression of 28 genes, many of which are known to be involved in epithelial–mesenchymal transition (EMT) and/or disease progression in several types of cancers, including but not limited to breast cancer, gastric cancer, cervical carcinoma, bladder cancer and thyroid cancer. This included several members of the S100 and IGFBP gene families. Taken together, these data support a role for PTC MSCs in PTC progression. Full article

The stroma of healthy pancreases contains various non-hematopoietic, non-endothelial mesenchymal cells. It is altered by chronic inflammation which in turn is a major contributor to the development of pancreatic adenocarcinoma (PDAC). In PDAC, the stroma plays a decisive and well-investigated role for tumor progression and therapy response. This review addresses the central role of stromal cells in the early inflammation-driven development of PDAC. It focuses on major subpopulations of pancreatic mesenchymal cells, i.e., fibroblasts, pancreatic stellate cells, and multipotent stroma cells, particularly their activation and functional alterations upon chronic inflammation including the development of different types of carcinoma-associated fibroblasts. In the second part, the current knowledge on the impact of activated stroma cells on acinar-to-ductal metaplasia and the transition to pancreatic intraepithelial neoplasia is summarized. Finally, putative strategies to target stroma cells and their signaling in early pancreatic carcinogenesis are reflected. In summary, the current data show that the activation of pancreatic stroma cells and the resulting fibrotic changes has pro- and anti-carcinogenetic effects but, overall, creates a carcinogenesis-promoting microenvironment. However, this is a dynamic process and the therapeutic targeting of specific pathways and cells requires in-depth knowledge of the molecular interplay of various cell types. Full article

Disruptions in uterine tissue function contribute to disorders such as endometriosis, adenomyosis, endometrial cancer, and fibroids, which all significantly impact health and fertility. Advances in transcriptomics, particularly single-cell RNA sequencing, have revolutionized uterine biological research by revealing the cellular heterogeneity and molecular mechanisms underlying disease states. Single-cell RNA sequencing and spatial transcriptomics have mapped endometrial and myometrial cellular landscapes, which helped to identify critical cell types, signaling pathways, and phase-specific dynamics. Said transcriptomic technologies also identified stromal and immune cell dysfunctions, such as fibroblast-to-myofibroblast transitions and impaired macrophage activity, which drive fibrosis, chronic inflammation, and lesion persistence in endometriosis. For endometrial cancer, scRNA-seq uncovered tumor microenvironmental complexities, identifying cancer-associated fibroblast subtypes and immune cell profiles contributing to progression and therapeutic resistance. Similarly, studies on adenomyosis highlighted disrupted signaling pathways, including Wnt and VEGF, and novel progenitor cell populations linked to tissue invasion and neuroinflammation, while single-cell approaches characterized smooth muscle and fibroblast subpopulations in uterine fibroids, elucidating their roles in extracellular matrix remodeling and signaling pathways like ERK and mTOR. Despite challenges such as scalability and reproducibility, single-cell transcriptomic approaches may have potential applications in biomarker discovery, therapeutic target identification, and personalized medicine in gynecological disorders. Full article

Immunosuppression is one key feature of mesenchymal stromal cells (MSCs) that has high expectations for therapeutic use. The influence of pro-inflammatory stimuli can modify the characteristics of MSCs and enhance immunosuppressive properties. The local postoperative environment contains cytokines, MSCs, and immune cells in high quantities, and their mutual influence is still unclear. Knowledge of in vivo processes is pivotal for potential therapeutic applications, and therefore, the aim of this study was to investigate the influence of wound fluid (WF) on the immunomodulatory potential of MSCs. CD4+ cells were co-cultured with native or WF-preconditioned MSCs for 5 days. CFSE staining revealed significant suppression of T cell proliferation after co-culture that was even more distinct in co-culture with WF-MSCs. The concentration of IDO-1, TGF-β1 and IFN-γ was higher while TNF-α was reduced in co-culture supernatants, indicating a transition to an anti-inflammatory milieu. In summary, the results provide evidence that the influence of WF alters the immunomodulatory potential of MSCs. These findings should serve as the basis for further investigations with a focus on T cell subpopulations. Full article

Constructing artificial tertiary lymphoid structures (TLSs) opens new avenues for advancing cancer immunotherapy and personalized medicine by creating controllable immune niches. Mesenchymal stromal cells (MSCs) offer an ideal stromal source for such constructs, given their potent immunomodulatory abilities and accessibility. In this study, we explored the potential of adipose-derived MSCs to adopt TLS-supportive phenotypes and facilitate lymphocyte organization. Single-cell RNA sequencing revealed a distinct subpopulation of MSCs expressing key fibroblastic reticular cell (FRC)-associated markers, including IL-7, PDPN, and IL-15, though lacking follicular dendritic cell (FDC) markers. TNF-α stimulation, but not LTα2β1, further enhanced FRC marker expression (IL-7, PDPN, and ICAM1). Notably, in 3D spheroid co-culture with lymphocytes, MSCs upregulated additional FRC markers, specifically CCL21. Upon implantation into adipose tissue, MSC-lymphocyte organoids maintained structural integrity and showed extensive T-cell infiltration and partial vascularization after 15 days in vivo, although organized B-cell follicles and FDC markers were still lacking. These findings highlight MSCs’ intrinsic ability to adopt an FRC-like phenotype that supports T-cell and HEV organization, suggesting that further optimization, including genetic modification, may be needed to achieve an FDC phenotype and replicate the full architectural and functional complexity of TLSs. Full article

Drug discovery is associated with high levels of compound elimination in all stages of development. The current practices for the pharmacokinetic testing of intestinal absorption combine Transwell^® inserts with the Caco-2 cell line and are associated with a wide range of limitations. The improvement of pharmacokinetic research relies on the development of more advanced in vitro intestinal constructs that better represent human native tissue and its response to drugs, providing greater predictive accuracy. Here, we present a humanized, bioengineered intestinal construct that recapitulates aspects of intestinal microanatomy. We present improved histotypic characteristics reminiscent of the human intestine, such as a reduction in transepithelial electrical resistance (TEER) and the formation of a robust basement membrane, which are contributed to in-part by a strong stromal foundation. We explore the link between stromal–epithelial crosstalk, paracrine communication, and the role of the keratinocyte growth factor (KGF) as a soluble mediator, underpinning the tissue-specific role of fibroblast subpopulations. Permeability studies adapted to a 96-well format allow for high throughput screening and demonstrate the role of the stromal compartment and tissue architecture on permeability and functionality, which is thought to be one of many factors responsible for unexpected drug outcomes using current approaches for pharmacokinetic testing. Full article

Corneal transparency and avascularity are essential for vision. The avascular cornea transitions into the vascularized conjunctiva at the limbus. Here, we explore a limbal stromal cell sub-population that expresses ABCB5 and has mesenchymal stem cell characteristics. Human primary corneal stromal cells were enriched for ABCB5 by using FACS sorting. ABCB5+ cells expressed the MSC markers CD90, CD73, and CD105. ABCB5+ but not ABCB5− cells from the same donor displayed evidence of pluripotency with a significantly higher colony-forming efficiency and the ability of trilineage differentiation (osteogenic, adipogenic, and chondrogenic). The ABCB5+ cell secretome demonstrated lower levels of the pro-inflammatory protein MIF (macrophage migration inhibitory factor) as well as of the pro-(lymph)angiogenic growth factors VEGFA and VEGFC, which correlated with reduced proliferation of Jurkat cells co-cultured with ABCB5+ cells and decreased proliferation of blood and lymphatic endothelial cells cultured in ABCB5+ cell-conditioned media. These data support the hypothesis that ABCB5+ limbal stromal cells are a putative MSC population with potential anti-inflammatory and anti-(lymph)angiogenic effects. The therapeutic modulation of ABCB5+ limbal stromal cells may prevent cornea neovascularization and inflammation and, if transplanted to other sites in the body, provide similar protective properties to other tissues. Full article

Limbal mesenchymal stromal cells (LMSCs) reside in the limbal niche, supporting corneal integrity and facilitating regeneration. While mesenchymal stem/stromal cells (MSCs) are used in regenerative therapies, there is limited knowledge about LMSC subpopulations and their characteristics. This study characterized human LMSC subpopulations through the flow cytometric assessment of fifteen cell surface markers, including MSC, wound healing, immune regulation, ASC, endothelial, and differentiation markers. Primary LMSCs were established from remnant human corneal transplant specimens and passaged eight times to observe changes during subculture. The results showed the consistent expression of typical MSC markers and distinct subpopulations with the passage-dependent expression of wound healing, immune regulation, and differentiation markers. High CD166 and CD248 expressions indicated a crucial role in ocular surface repair. CD29 expression suggested an immunoregulatory role. Comparable pigment-epithelial-derived factor (PEDF) expression supported anti-inflammatory and anti-angiogenic roles. Sustained CD201 expression indicated maintained differentiation capability, while VEGFR2 expression suggested potential endothelial differentiation. LMSCs showed higher VEGF expression than fibroblasts and endothelial cells, suggesting a potential contribution to ocular surface regeneration through the modulation of angiogenesis and inflammation. These findings highlight the heterogeneity and multipotent potential of LMSC subpopulations during in vitro expansion, informing the development of standardized protocols for regenerative therapies and improving treatments for ocular surface disorders. Full article

A T-cell-independent (TI) pathway activated by microbiota results in the generation of low-affinity homeostatic IgA with a critical role in intestinal homeostasis. Moderate aerobic exercise (MAE) provides a beneficial impact on intestinal immunity, but the action of MAE on TI-IgA generation under senescence conditions is unknown. This study aimed to determine the effects of long-term MAE on TI-IgA production in young (3 month old) BALB/c mice exercised until adulthood (6 months) or aging (24 months). Lamina propria (LP) from the small intestine was obtained to determine B cell and plasma cell sub-populations by flow cytometry and molecular factors related to class switch recombination [Thymic Stromal Lymphopoietin (TSLP), A Proliferation-Inducing Ligand (APRIL), B Cell Activating Factor (BAFF), inducible nitric oxide synthase (iNOS), and retinal dehydrogenase (RDH)] and the synthesis of IgA [α-chain, interleukin (IL)-6, IL-21, and Growth Factor-β (TGF-β)]; and epithelial cells evaluated IgA transitosis [polymeric immunoglobulin receptor (pIgR), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-4] by the RT-qPCR technique. The results were compared with data obtained from sedentary age-matched mice. Statistical analysis was computed with ANOVA, and p < 0.05 was considered to be a statistically significant difference. Under senescence conditions, MAE promoted the B cell and IgA+ B cells and APRIL, which may improve the intestinal response and ameliorate the inflammatory environment associated presumably with the downmodulation of pro-inflammatory mediators involved in the upmodulation of pIgR expression. Data suggested that MAE improved IgA and downmodulate the cytokine pro-inflammatory expression favoring homeostatic conditions in aging. Full article