Changes In Microenvironment Research Articles

1336P Clinical outcomes, toxic effect, and changes of immune microenvironment of drug-eluting beads bronchial artery chemoembolization (DEB-BACE) combined with immunotherapy in the treatment of elder patients with non-small cell lung cancer

1336P Clinical outcomes, toxic effect, and changes of immune microenvironment of drug-eluting beads bronchial artery chemoembolization (DEB-BACE) combined with immunotherapy in the treatment of elder patients with non-small cell lung cancer

High doses of radiation cause cochlear immunological stress and sensorineural hearing loss

Radiotherapy is a crucial treatment for head and neck malignancies, but it can sometimes cause sensorineural hearing loss (SNHL). Changes in the immune microenvironment and sensory neuroepithelium of the inner ear after radiation exposure remain poorly understood. This study investigated cochlear morphology and macrophages in the inner ear after high-dose irradiation. The heads of heterozygous 8-week-old Cx3cr1GFP/+ male mice were irradiated with 30Gy of X-rays and biological samples were collected on days 1, 7, and 10 after irradiation. Auditory brainstem responses were used to assess auditory function in the mice. Changes in basilar membrane hair cells, spiral ganglion neurons (SGN), and inner ear macrophages were observed using hematoxylin-eosin (HE) staining and immunofluorescence staining. The expression of inflammatory mediators in the inner ear was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) in cochlear tissue. The results showed no significant hair cell loss after a single high dose of radiation. However, the mice developed pantonal hearing loss on day 10 when HE staining revealed SGN atrophy and immunofluorescence showed decreased neurofilament expression. The number of macrophages in the inner ear reduced over time. RT-qPCR showed that cochlear inflammatory factors and chemokines were briefly upregulated on day 1st after irradiation and then decreased over time. In conclusion, high-dose irradiation causes acute SNHL that is not associated with hair cell loss and may be related to SGN changes. Radiation-induced SNHL is associated with a reduction in cochlear macrophages and changes in the immune microenvironment, but the relationship between the two remains to be investigated.

AMPK, a hub for the microenvironmental regulation of bone homeostasis and diseases.

AMP-activated protein kinase (AMPK), a crucial regulatory kinase, monitors energy levels, conserving ATP and boosting synthesis in low-nutrition, low-energy states. Its sensitivity links microenvironmental changes to cellular responses. As the primary support structure and endocrine organ, the maintenance, and repair of bones are closely associated with the microenvironment. While a series of studies have explored the effects of specific microenvironments on bone, there is lack of angles to comprehensively evaluate the interactions between microenvironment and bone cells, especially for bone marrow mesenchymal stem cells (BMMSCs) which mediate the differentiation of osteogenic lineage. It is noteworthy that accumulating evidence has indicated that AMPK may serve as a hub between BMMSCs and microenvironment factors, thus providing a new perspective for us to understand the biology and pathophysiology of stem cells and bone. In this review, we emphasize AMPK's pivotal role in bone microenvironment modulation via ATP, inflammation, reactive oxygen species (ROS), calcium, and glucose, particularly in BMMSCs. We further explore the use of AMPK-activating drugs in the context of osteoarthritis and osteoporosis. Moreover, building upon the foundation of AMPK, we elucidate a viewpoint that facilitates a comprehensive understanding of the dynamic relationship between the microenvironment and bone homeostasis, offering valuable insights for prospective investigations into stem cell biology and the treatment of bone diseases.

Shifting the paradigm in personalized cancer care through next-generation therapeutics and computational pathology.

The incorporation of novel therapeutic agents such as antibody-drug conjugates, radio-conjugates, T-cell engagers, and chimeric antigen receptor cell therapies represents a paradigm shift in oncology. Cell-surface target quantification, quantitative assessment of receptor internalization, and changes in the tumor microenvironment (TME) are essential variables in the development of biomarkers for patient selection and therapeutic response. Assessing these parameters requires capabilities that transcend those of traditional biomarker approaches based on immunohistochemistry, in situ hybridization and/or sequencing assays. Computational pathology is emerging as a transformative solution in this new therapeutic landscape, enabling detailed assessment of not only target presence, expression levels, and intra-tumor distribution but also of additional phenotypic features of tumor cells and their surrounding TME. Here, we delineate the pivotal role of computational pathology in enhancing the efficacy and specificity of these advanced therapeutics, underscoring the integration of novel artificial intelligence models that promise to revolutionize biomarker discovery and drug development.

Analysis of metastasis‑related risk factors and clinical relevance in adult soft‑tissue sarcoma.

Metastasis occurs in nearly 50% of cases of adult soft-tissue sarcoma (ASTS), leading to a dismal prognosis, with a 2-year survival rate of ~30%. Consequently, a prognostic model that incorporates metastatic characteristics may be instrumental in predicting survival time and in crafting optimal personalized therapeutic strategies for patients with ASTS. In the present study, a prognostic prediction model for ASTS was developed by examining genes that are differentially expressed between non-metastatic and metastatic patients in the Gene Expression Omnibus dataset. The prognostic model, which includes five featured genes [actin γ2 (ACTG2), apolipoprotein D, coatomer protein complex subunit γ2 imprinted transcript 1, collagen type VI α6 chain and osteomodulin], was further validated in patients with ASTS from the Cancer Genome Atlas dataset. Based on these five-gene signatures, patients were categorized into high- and low-risk groups. Functional and pathway analyses revealed disparities in stemness, extracellular matrix and cell adhesion-related pathways between the two risk groups, particularly noting the activation of the PI3K-Akt pathway in high-risk cases. Analysis of immune infiltration also revealed variations in immune microenvironment changes between the two risk groups. Immunohistochemical staining substantiated the prognostic significance of these gene signatures in a specific sarcoma subtype. Additionally, wound-healing and Transwell assays demonstrated that inhibition of ACTG2 by shRNAs curbed cell migration and invasion in a sarcoma HOS cell line, underscoring its role in sarcoma metastasis. In conclusion, the present study successfully developed and validated a metastasis-based prognosis prediction model. This model not only reliably forecasts the survival of patients with ASTS, but also may pave the way for further investigation into the processes underlying sarcoma metastasis, ultimately aiding in the design of tailored therapeutic regimens.

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer

How prostate cancer cells and their precursors mediate changes in the tumor microenvironment (TME) to drive prostate cancer progression is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we perform extensive single-cell RNA-sequencing (scRNA-seq) and molecular pathology of the comparative biology between human prostate cancer and key stages in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues reveal that cancer cell-intrinsic activation of MYC signaling is a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Cell communication network and pathway analyses in GEMMs show that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogram the TME during carcinogenesis, leading to a convergence of cell state alterations in neighboring epithelial, immune, and fibroblast cell types that parallel key findings in human prostate cancer.

Profiling biomanufactured extracellular vesicles of human forebrain spheroids in a Vertical-Wheel Bioreactor.

Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell-free therapies in various diseases, including stroke. However, because of the significant amount of EVs needed in preclinical and clinical trials, EV applicationis still challenging. Vertical-Wheel Bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress. In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3D aggregates and on Synthemax II microcarriers in VWBRs were investigated with static aggregate culture as a control. The spheroids were characterized by metabolite and transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. The EV cargo was analyzed using proteomics and miRNA sequencing. The in vitro functional assays of an oxygen and glucose-deprived stroke model were conducted. Proof of concept in vivo study was performed, too. Human forebrain spheroid differentiated on microcarriers showed a higher growth rate than 3D aggregates. Microcarrier culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes. EVs from the three culture conditions showed no differences in size, but the yields from high to low were microcarrier cultures, dynamic aggregates, and static aggregates. The cargo is enriched with proteins (proteomics) and miRNAs (miRNA-seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. Human forebrain spheroid EVs demonstrated the ability to improve recovery in an in vitro stroke model and in vivo. Human forebrain spheroid differentiation in VWBR significantly increased the EV yields (up to 240-750 fold) and EV biogenesis compared to static differentiation due to the dynamic microenvironment and metabolism change. The biomanufactured EVs from VWBRs have exosomal characteristics and more therapeutic cargo and are functional in in vitro assays, which paves the way for future in vivo stroke studies.

Integrating transcriptomics and proteomics to analyze the immune microenvironment of cytomegalovirus associated ulcerative colitis and identify relevant biomarkers

BackgroundIn recent years, significant morbidity and mortality in patients with severe inflammatory bowel disease (IBD) and cytomegalovirus (CMV) have drawn considerable attention to the status of CMV infection in the intestinal mucosa of IBD patients and its role in disease progression. However, there is currently no high-throughput sequencing data for ulcerative colitis patients with CMV infection (CMV + UC), and the immune microenvironment in CMV + UC patients have yet to be explored.MethodThe xCell algorithm was used for evaluate the immune microenvironment of CMV + UC patients. Then, WGCNA analysis was explored to obtain the co-expression modules between abnormal immune cells and gene level or protein level. Next, three machine learning approach include Random Forest, SVM-rfe, and Lasso were used to filter candidate biomarkers. Finally, Best Subset Selection algorithms was performed to construct the diagnostic model.ResultsIn this study, we performed transcriptomic and proteomic sequencing on CMV + UC patients to establish a comprehensive immune microenvironment profile and found 11 specific abnormal immune cells in CMV + UC group. After using multi-omics integration algorithms, we identified seven co-expression gene modules and five co-expression protein modules. Subsequently, we utilized various machine learning algorithms to identify key biomarkers with diagnostic efficacy and constructed an early diagnostic model. We identified a total of eight biomarkers (PPP1R12B, CIRBP, CSNK2A2, DNAJB11, PIK3R4, RRBP1, STX5, TMEM214) that play crucial roles in the immune microenvironment of CMV + UC and exhibit superior diagnostic performance for CMV + UC.ConclusionThis 8 biomarkers model offers a new paradigm for the diagnosis and treatment of IBD patients post-CMV infection. Further research into this model will be significant for understanding the changes in the host immune microenvironment following CMV infection.

19Fluorine-MRI Based Longitudinal Immuno-Microenvironment-Monitoring for Pancreatic Cancer.

Pancreatic cancer has a poor prognosis. Targeting Kirsten Rat Sarcoma (KRAS) mutation and its related pathways may enhance immunotherapy efficacy. While in vivo monitoring of therapeutic response and immune cell migration remains challenging, Fluorine-19 MRI (19F MRI) may allow noninvasive longitudinal imaging of immune cells. Evaluating the potential of 19F MRI for monitoring changes in the tumor immune microenvironment, in response to combined SHP2/MEK inhibition. Pre-clinical animal study. Murine genetically engineered pancreatic cancer model (N = 20, both sexes). 9.4-T, two-dimensional multi-slice Rapid Acquisition with Relaxation Enhancement sequence. Intravenous injection of 19F-perfluorocarbon (PFC) nanoparticles. Upon tumor detection by conventional 1H MRI screening, 19F MRI was performed in mice 24 hours after PFC nanoparticle administration. Animals were randomly assigned to four treatment groups: allosteric Src-homology-2-containing protein tyrosine phosphatase 2 (SHP2) inhibitor SHP099, the mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor Trametinib, the combination of both, or a vehicle control (4 to 6 mice each group), administered every other day per oral gavage. 1H and 19F MRI was repeated 7 days and 14 days later. Pancreatic immune cell infiltrates were analyzed by flow cytometry and multiplex immunohistofluorescence (mIHF) upon sacrifice. Independent t-tests and one-way analysis of variance. 19F MRI revealed continuous decrease of PFC-signals in tumors from vehicle controls (100%, 80%, and 74% on days 0, 7, and 14, respectively), contrasting with stable or increasing signals under KRAS-pathway-directed treatment. MEK inhibition showed 100%, 152%, and 84% and dual SHP2/MEK1/2 inhibition demonstrated signals of 100%, 134%, and 100% on days 0, 7, 14, respectively. mIHF analyses indicated CD11b+ macrophages/monocytes as primary contributors to the observed 19F MRI signal differences. 19F MRI might provide non-invasive longitudinal estimates for abundance and spatial distribution of CD11b+ macrophages/monocytes in pancreatic cancer. 1 TECHNICAL EFFICACY: Stage 2.

B-cell depletion limits HTLV-1-infected T-cell expansion and ameliorate HTLV-1-associated myelopathy.

Human T-cell leukemia virus type 1-associated myelopathy (HAM) is a chronic, progressive, inflammatory disease with unclear pathogenesis and no effective treatments. We aimed to investigate a novel mechanistic theory and treat HAM patients with rituximab, which can deplete CD20+ B lymphocytes in circulation. Single-cell RNA sequencing (scRNA-seq) data was analyzed to identify HTLV-1-associated B cells and their effect on Tcells. An observational analysis of our HAM cohort was conducted to elucidate changes in the immunological microenvironment of these patients. Peripheral blood mononuclear cells (PBMC) from HAM patients were isolated to explore the efficacy of B cell depletion in vitro. To assess the effect of B-cell depletion on HAM patients, eligible participants in our cohort received rituximab therapy (NCT04004819). ScRNA-seq results suggest a significant effect of HTLV-1-associated B cells on Tcells. Additionally, HTLV-1 was found to infect B cells and depletion of B cells inhibited the proliferation of Tcells. Number of B cells in HAM patients had positive correlation with the proviral load and infected cell counts. Depletion of B cells led to a reduction in HTLV-1 proviral load in vitro. Furthermore, in clinical trial, 14 HAM patients were enrolled. Three patients (21.4%) who received rituximab failed to achieve remission, compared to 24 (85.7%) patients received any other therapy that failed to achieve remission. With a low level of circulating B cells, the proportion of Ki67-positive cells in CD4+ Tcells fell. This study provided evidence that depleting B-lymphocytes is an innovative strategy for treating patients with HAM and broadens the understanding of the role of B cells in infectious immunity.

Design of a liposome casein hydrogel as an efficient front-end homeostatic anthocyanin loading system

Proteins have been studied and applied to improve the stability of anthocyanins (ACNs), but the changes in the pH microenvironment during the preparation of steady-state systems are often ignored, and more attention is given to the stability of the system after preparation. In this study, we propose the “anthocyanin front-end homeostasis strategy”, which involves designing a system can protect anthocyanins under acidic conditions so that more anthocyanin prototypes can be loaded inside the protein. Anthocyanins are encapsulated in liposomes (Lip) at pH 3.0 and combined with casein methacrylate (CSMA) to form Anthocyanin-loaded liposomes/CSMA hydrogel (Lip@ACNs/CSMA), with good physical properties and good blood compatibility. The system increased the hydrogen peroxide scavenging capacity by 1.16 mg Vc equiv./mg ACNs and the cellular antioxidant activity by 17.55 μM quercetin/100 mg ACNs, the photo and thermal storage stability increased by 36.50 % and 30.71 %, the digestive rate increased by 17.50 %, and the biological availability increased by 0.0049 mg/mL. This study designed a liposome casein hydrogel as an efficient front-end homeostatic anthocyanin loading system and provided a new approach for improving the stability of anthocyanins.

The Effects of Different Developmental Stages on Bone Regeneration of Periodontal Ligament Stem Cells and Periodontal Ligament Cell Sheets In Vitro and Vivo.

Periodontal ligament stem cells (PDLSCs) have broad applications in tissue engineering and regeneration. However, the function of PDLSCs changes in different microenvironments. This study aimed to explore the effects of different developmental stages on the biological characteristics of PDLSCs. Here, PDLSCs were successfully cultured from the periodontal tissues of various groups, including a group with immature roots, a young group with mature roots, and an aging group with mature roots. By comparing the results of the three experimental groups, we found that the donors with immature roots exhibited the best proliferative ability and osteogenic differentiation, while the aging group demonstrated the worst proliferation. The trend for adipogenic differentiation was the opposite to that for osteogenic differentiation. The cell sheet and in vivo experiments revealed that in the immature root group, the cells produced more extracellular matrix (ECM) and new bone and better absorbed the implant materials. These results indicate that PDLSCs perform various functions at different stages of development. In clinical applications of PDLSCs for periodontal regeneration, donors with incompletely developed roots have stronger biological characteristics.

Inhibition of α-amylase activity by quercetin via multi-spectroscopic and molecular docking approaches

The quercetin-induced inhibition of α-amylase activity was comprehensively studied using various spectroscopic methods, including UV–Visible, fluorescence, and Fourier-transform infrared spectroscopy. Quercetin acted as a mixed-type inhibitor, and its binding altered the kinetic properties of α-amylase. Quercetin exhibited static binding with α-amylase, as evidenced by a higher Stern-Volmer constant (KSV) at 298 K (9.56 ± 0.63 × 103 L/mol) compared to the KSV value at 318 K (5.56 ± 0.48 × 103 L/mol). Additionally, quercetin induced conformational changes in the hydrophobic micro-environment surrounding the tryptophan and tyrosine residues of α-amylase, as indicated by fluorescence spectroscopy. Hydrophobic interactions primarily drove the spontaneous binding between α-amylase and quercetin from thermodynamic data. Molecular docking studies supported the role of hydrogen bonds (Asp197 and Gln63) and hydrophobic interactions (with active site residues Ala198, Leu165, and Trp59) in the complex formation. These findings provide a detailed understanding of how quercetin inhibits α-amylase activity, reinforcing its potential as a glycemic controller in food formulations.

A New Perspective on Stroke Research: Unraveling the Role of Brain Oxygen Dynamics in Stroke Pathophysiology.

Stroke, a leading cause of death and disability, often results from ischemic events that cut off the brain blood flow, leading to neuron death. Despite treatment advancements, survivors frequently endure lasting impairments. A key focus is the ischemic penumbra, the area around the stroke that could potentially recover with prompt oxygenation; yet its monitoring is complex. Recent progress in bioluminescence-based oxygen sensing, particularly through the Green enhanced Nano-lantern (GeNL), offers unprecedented views of oxygen fluctuations in vivo. Utilized in awake mice, GeNL has uncovered hypoxic pockets within the cerebral cortex, revealing the brain's oxygen environment as a dynamic landscape influenced by physiological states and behaviors like locomotion and wakefulness. These findings illuminate the complexity of oxygen dynamics and suggest the potential impact of hypoxic pockets on ischemic injury and recovery, challenging existing paradigms and highlighting the importance of microenvironmental oxygen control in stroke resilience. This review examines the implications of these novel findings for stroke research, emphasizing the criticality of understanding pre-existing oxygen dynamics for addressing brain ischemia. The presence of hypoxic pockets in non-stroke conditions indicates a more intricate hypoxic scenario in ischemic brains, suggesting strategies to alleviate hypoxia could lead to more effective treatments and rehabilitation. By bridging gaps in our knowledge, especially concerning microenvironmental changes post-stroke, and leveraging new technologies like GeNL, we can pave the way for therapeutic innovations that significantly enhance outcomes for stroke survivors, promising a future where an understanding of cerebral oxygenation dynamics profoundly informs stroke therapy.

Interaction between Halogenated Phenols and Thyroxine‐Binding Protein: A Multispectral and Computational Approach

AbstractThe aim of this study was to understand the interaction between transthyretin (TTR) and halogenated phenols (HPs) including 2,4‐dichlorophenol (DCP), 2,4,6‐trichlorophenol (TCP), pentachlorophenol (PCP), 2‐bromophenol (2‐BP), 2,4‐dibromophenol (DBP) and 2,4,6‐tribromophenol (TBP). Computer simulation and multi‐spectroscopy techniques were used to investigate the interaction. The fluorescence of TTR was found to be quenched through static quenching and non‐radiative energy transfer by HPs, suggesting that the binding process between HPs and TTR was primarily influenced by van der Waals forces and hydrogen bonds. ANS substitution experiments indicated that HPs interacted with TTR at the T4 binding site. Three‐dimensional fluorescence spectra and Fourier transform infrared spectroscopy were used to investigate the conformational and microenvironmental changes of TTR resulting from the interactions with HPs. Additionally, molecular docking and reduced density gradient analysis were used to identify the specific residues and binding forces involved in the interaction between HPs and TTR binding sites. The findings were subsequently validated through quantum chemical analysis. This research integrates state‐of‐the‐art multispectral analytical methodologies with sophisticated computational modeling to introduce an innovative approach for dissecting the interactions between halogenated phenols (HPs) and transthyretin (TTR). This methodology offers a novel avenue for investigating the molecular interplay between HPs and TTR, thereby providing critical insights that are instrumental in assessing the latent toxicity of these compounds.

Integrated omics characterization reveals reduced cancer indicators and elevated inflammatory factors after thermal ablation in non-small cell lung cancer patients

BackgroundThermal ablation is a minimally invasive treatment for non-small cell lung cancer (NSCLC). Aside from causing an immediate direct tumour cell injury, the effects of thermal ablation on the internal microenvironment are unknown. This study aimed to investigate the effects of thermal ablation on the plasma internal environment in patients with NSCLC.Methods128 plasma samples were collected from 48 NSCLC (pre [LC] and after thermal ablation [LC-T]) patients and 32 healthy controls (HCs). Olink proteomics and metabolomics were utilized to construct an integrated landscape of the cancer-related immune and inflammatory responses after ablation.ResultsCompared with HCs, LC patients exhibited 58 differentially expressed proteins (DEPs) and 479 differentially expressed metabolites (DEMs), which might participate in tumour progression and metastasis. Moreover, 75 DEPs were identified among the HC, LC, and LC-T groups. Forty-eight highly expressed DEPs (eg, programmed death-ligand 1 [PD-L1]) in the LC group were found to be downregulated after thermal ablation. These DEPs had significant impacts on pathways such as angiogenesis, immune checkpoint blockade, and pro-tumour chemotaxis. Metabolites involved in tumour cell survival were associated with these proteins at the expression and functional levels. In contrast, 19 elevated proteins (eg, interleukin [IL]-6) were identified after thermal ablation. These proteins were mainly associated with inflammatory response pathways (NF-κB signalling and tumour necrosis factor signalling) and immune cell activation.ConclusionsThermal ablation-induced changes in the host plasma microenvironment contribute to anti-tumour immunity in NSCLC, offering new insights into tumour ablation combined with immunotherapy.Trial registration This study was registered on the Chinese Clinical Trial Registry (https://www.chictr.org.cn/index.html). ID: ChiCTR2300076517. Registration Date: 2023-10-11.

Long-term tracking of lysosomal dynamics with highly stable fluorescent probe

Monitoring lysosomal dynamics in real-time, especially in vivo, poses significant challenges due to the complex and dynamic nature of cellular environments. It is extremely important to construct fluorescent probes with high stability for imaging lysosomes to minimize interference from other cellular components, in order to ensure prolonged imaging. A fluorescent probe (PDB) has been proposed for targeting lysosomes, which was less affected to changes in the cellular microenvironment (such as pH, viscosity and polarity). PDB can be easily prepared by 4′-piperazinoacetophenone and 2-(4-diethylamino)-2-hydroxybenzoyl) benzoicacid, containing a piperazine group for labeling and imaging lysosomes and the high pKa value (∼9.35) allowed PDB to efficiently track lysosomes. The emission wavelength of PDB in aqueous solution was 634 nm (λex = 572 nm, Фf = 0.11). The dynamic process of lysosome induced by starvation and rapamycin was successfully explored by fluorescence imaging. Compared with the commercially available Lyso-Tracker green, the high photostability fluorescent probe can ensure 3D high-fidelity tracking and resist photobleaching. Therefore, PDB, unaffected by the cell microenvironment, successfully achieved long-term tracking of lysosomal movement, even enabling imaging in tumor-bearing mice over 11 days. The strong fluorescence signal, high stability, and long-term tracking capability indicate that PDB has tremendous potential in monitoring biological processes.

Extracellular adenosine oppositely regulates the purinome machinery in glioblastoma and mesenchymal stem cells.

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed-back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB-conditioned medium or co-cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell-based strategies using MSCs.

Simultaneous Mapping of the Nanoscale Organization and Redox State of Extracellular Space in Living Brain Tissue.

The spatial organization characteristics and redox status of the extracellular space (ECS) are crucial in the development of brain diseases. However, it remains a challenge to simultaneously capture dynamic changes in microstructural features and redox states at the submicron level within the ECS. Here, we developed a reversible glutathione (GSH)-responsive nanoprobe (RGN) for mapping the spatial organization features and redox status of the ECS in brain tissues with nanoscale resolution. The RGN is composed of polymer nanoparticles modified with GSH-responsive molecules and amino-functionalized methoxypoly(ethylene glycol), which exhibit exceptional single-particle brightness and excellent free diffusion capability in the ECS of brain tissues. Tracking single RGNs in acute brain slices allowed us to dynamically map spatial organizational features and redox levels within the ECS of brain tissues in disease models. This provides a powerful super-resolution imaging method that offers a potential opportunity to study the dynamic changes in the ECS microenvironment and to understand the physiological and pathological roles of the ECS in vivo.

Prostate cancer–induced endothelial-cell-to-osteoblast transition drives immunosuppression in the bone–tumor microenvironment through Wnt pathway–induced M2 macrophage polarization

Immune checkpoint therapy has limited efficacy for patients with bone-metastatic castration-resistant prostate cancer (bmCRPC). To improve immunotherapy for bmCRPC, we aimed to identify the mechanism of bmCRPC-induced changes in the immune microenvironment. Among bmCRPC patients, higher levels of a 32-gene M2-like macrophage signature in bone metastasis samples correlated with shorter overall survival. Immunohistochemistry showed that CD206-positive (CD206+) macrophages were enriched in bmCRPC bone biopsy specimens compared with primary tumors or lymph node metastases. In preclinical osteogenic prostate cancer (Pca) xenograft models, CD206+ macrophages were recruited to areas with tumor-induced bone. RNA sequencing (RNAseq) analysis showed higher expression of an M2-like gene signature, with activated canonical and noncanonical Wnt pathways, in tumor-associated macrophages isolated from osteogenic tumors (bone-TAMs) than in TAMs isolated from nonosteogenic tumors (ctrl-TAMs). Mechanistic studies showed that endothelial cells (ECs) that had undergone EC-to-osteoblast (EC-to-OSB) transition, the precursors of tumor-induced OSBs, produced paracrine factors, including Wnts, CXCL14, and lysyl oxidase, which induced M2 polarization and recruited M2-like TAMs to the bone-tumor microenvironment (bone-TME). Bone-TAMs suppressed CD8+ T cells' proliferation and cytolytic activity, and these effects were partially reversed by treating bone-TAMs with Wnt inhibitors. Genetic or pharmacological inhibition of Pca-induced EC-to-OSB transition reduced the levels of M2-like macrophages in osteogenic tumors. Our study demonstrates that Pca-induced EC-to-OSB transition drives immunosuppression in the bone-TME, suggesting that therapies that reduce Pca-induced bone formation may improve immunotherapeutic outcomes for bmCRPC.