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Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.
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Células Dendríticas Foliculares/imunologia , Fibroblastos/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Idoso , Animais , Apoptose/genética , Apoptose/imunologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Dendríticas Foliculares/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Humanos , Imunidade Celular/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfonodos/citologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Análise de Célula Única , Células Estromais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Achilles tendon rupture is a common sports-related injury. Even with advanced clinical treatments, many patients suffer from long-term pain and functional deficits. These unsatisfactory outcomes result primarily from an imbalanced injury response with excessive inflammation and inadequate tendon regeneration. Prior studies showed that extracellular vesicles from inflammation-primed adipose-derived stem cells (iEVs) can attenuate early tendon inflammatory response to injury. It remains to be determined if iEVs can both reduce inflammation and promote regeneration in the later phases of tendon healing and the underlying mechanism. Therefore, this study investigated the mechanistic roles of iEVs in regulating tendon injury response using a mouse Achilles tendon injury and repair model in vivo and iEV-macrophage and iEV-tendon cell coculture models in vitro. Results showed that iEVs promoted tendon anti-inflammatory gene expression and reduced mononuclear cell accumulation to the injury site in the remodeling phase of healing. iEVs also increased collagen deposition in the injury center and promoted tendon structural recovery. Accordingly, mice treated with iEVs showed less peritendinous scar formation, much lower incidence of postoperative tendon gap or rupture, and faster functional recovery compared to untreated mice. Further in vitro studies revealed that iEVs both inhibited macrophage M1 polarization and increased tendon cell proliferation and collagen production. The iEV effects were partially mediated by miR-147-3p, which blocked the toll-like receptor 4/NF-κB signaling pathway that activated the M1 phenotype of macrophages. The combined results demonstrate that iEVs are a promising therapeutic agent that can enhance tendon repair by attenuating inflammation and promoting intrinsic healing.
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Tendão do Calcâneo , Vesículas Extracelulares , Células-Tronco Mesenquimais , Traumatismos dos Tendões , Humanos , Tendão do Calcâneo/lesões , Células-Tronco Mesenquimais/metabolismo , Cicatrização/fisiologia , Traumatismos dos Tendões/cirurgia , Vesículas Extracelulares/metabolismo , Colágeno , InflamaçãoRESUMO
Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server-based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.
A key concept in biology is that the structure of tissues, cells and their components (cell organelles) often relates to their function. With electron microscopy (EM), it is possible to reveal this structure with nanometre resolution and therefore infer about its function. Electron microscopy of tissues knows a long history of method development, starting in the 1940s. Method development has largely determined the possibilities and scope of electron microscopy. In the 2000s, innovative techniques were developed that allowed routine imaging of tissues in 3D with a higher degree of automation. Nevertheless, conventional electron microscopy techniques remain unsuited for imaging of tissue with nanometre resolution on a millimetre scale because of their low inherent throughput. Here we analyse trends in volume electron microscopy (EM of tissues in 3D) by reviewing the application, acquisition parameters and data information from over 100 publications in the field. We see an expansion of interest from the conventional applications in neuroscience to other fields, such as cell biology. Additionally, the size of data sets is growing rapidly. From here, we review in detail how certain developments in methodology from the past 10 years have tried to overcome the low acquisition throughput of electron microscopes, by making these techniques more robust during long acquisitions, but also much faster by parallelisation. We find that these new developments have big implications for sample preparation, processing and analysis of the images and data management. We therefore also describe the new developments in these separate domains. We illustrate how novel sample preparation protocols have been developed specifically for larger volumes, how the introduction of machine learning has accelerated automated segmentation of volume EM data and that there is an ongoing transition from local to remote data storage and management. We also touch upon the tools that researchers use to analyse and annotate EM data. We conclude that the potential of volume EM remains high and the new developments open up possibilities for novel biological studies. We promote the sharing of resources and tools between researchers and institutions to maximise the potential from the new developments in volume electron microscopy.
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Processamento de Imagem Assistida por Computador , Manejo de Espécimes , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Microscopia Eletrônica , Proteínas , Manejo de Espécimes/métodosRESUMO
The authors present the application of a retarding field between the electron objective lens and sample in an integrated fluorescence and electron microscope. The retarding field enhances signal collection and signal strength in the electron microscope. This is beneficial for samples prepared for integrated fluorescence and electron microscopy as the amount of staining material added to enhance electron microscopy signal is typically lower compared to conventional samples in order to preserve fluorescence. We demonstrate signal enhancement through the applied retarding field for both 80-nm post-embedding immunolabeled sections and 100-nm in-resin preserved fluorescence sections. Moreover, we show that tuning the electron landing energy particularly improves imaging conditions for ultra-thin (50 nm) sections, where optimization of both retarding field and interaction volume contribute to the signal improvement. Finally, we show that our integrated retarding field setup allows landing energies down to a few electron volts with 0.3 eV dispersion, which opens new prospects for assessing electron beam induced damage by in situ quantification of the observed bleaching of the fluorescence following irradiation.
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Optically pumped molecular gas amplifiers having a gain medium contained in a hollow-core optical fiber are investigated with numerical modeling to understand the primary physical processes that affect amplifier output and efficiency. A comparison of computational results with experimental measurements of incident pump, absorbed pump, and emitted mid-IR from a pulsed, acetylene-filled, hollow-core fiber amplifier [ Opt. Exp.25, 13351 (2017)] is used to explore the effects of various physical processes on pulsed amplifier operation. Single frequency, one-dimensional, time-dependent models are shown to align with experimentally measured lasing thresholds and ratios of absorbed pump to emitted laser energy but significantly over predict the amount of incident pump energy that is absorbed. A two-dimensional, time-dependent model that assumes Gaussian spectral and radial intensity profiles for the pump is developed and shows an improved ability to capture pump absorption. Results indicate that 1D, time-dependent models have utility in guiding experiments but the significant influence of the pump and laser propagation modes and the pump spectral characteristics on efficiency, threshold, and signal output must be explicitly included in high-fidelity high-power modeling.
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Recent advances in electron microscopy techniques have led to a significant scale up in volumetric imaging of biological tissue. The throughput of electron microscopes, however, remains a limiting factor for the volume that can be imaged in high resolution within reasonable time. Faster detection methods will improve throughput. Here, we have characterized and benchmarked a novel detection technique for scanning electron microscopy: optical scanning transmission electron microscopy (OSTEM). A qualitative and quantitative comparison was performed between OSTEM, secondary and backscattered electron detection and annular dark field detection in scanning transmission electron microscopy. Our analysis shows that OSTEM produces images similar to backscattered electron detection in terms of contrast, resolution and signal-to-noise ratio. OSTEM can complement large scale imaging with (scanning) transmission electron microscopy and has the potential to speed up imaging in single-beam scanning electron microscope.
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Cancer development and progression are generally associated with dysregulation of gene expression, often resulting from changes in transcription factor (TF) sequence or expression. Identifying key TFs involved in cancer gene regulation provides a framework for potential new therapeutics. This study presents a large-scale cancer gene TF-DNA interaction network as well as an extensive promoter clone resource for future studies. Most highly connected TFs do not show a preference for binding to promoters of genes associated with either good or poor cancer prognosis, suggesting that emerging strategies aimed at shifting gene expression balance between these two prognostic groups may be inherently complex. However, we identified potential for oncogene targeted therapeutics, with half of the tested oncogenes being potentially repressed by influencing specific activator or bifunctional TFs. Finally, we investigate the role of intrinsically disordered regions within the key cancer-related TF estrogen receptor É (ESR1) on DNA binding and transcriptional activity, and found that these regions can have complex trade-offs in TF function. Altogether, our study not only broadens our knowledge of TFs involved in the cancer gene regulatory network but also provides a valuable resource for future studies, laying a foundation for potential therapeutic strategies targeting TFs in cancer.
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Cancer development and progression are generally associated with gene dysregulation, often resulting from changes in the transcription factor (TF) sequence or expression. Identifying key TFs involved in cancer gene regulation provides a framework for potential new therapeutics. This study presents a large-scale cancer gene TF-DNA interaction network, as well as an extensive promoter clone resource for future studies. Highly connected TFs bind to promoters of genes associated with either good or poor cancer prognosis, suggesting that strategies aimed at shifting gene expression balance between these two prognostic groups may be inherently complex. However, we identified potential for oncogene-targeted therapeutics, with half of the tested oncogenes being potentially repressed by influencing specific activators or bifunctional TFs. Finally, we investigate the role of intrinsically disordered regions within the key cancer-related TF ESR1 in DNA binding and transcriptional activity, and found that these regions can have complex trade-offs in TF function. Altogether, our study broadens our knowledge of the TFs involved in cancer gene regulation and provides a valuable resource for future studies and therapeutics.
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DNA , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias , Ligação Proteica , Fatores de Transcrição , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , DNA/metabolismo , DNA/genética , Regiões Promotoras Genéticas/genética , Oncogenes/genética , Prognóstico , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Biologia Computacional/métodosRESUMO
Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.
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Following immunization, lymph nodes dynamically expand and contract. The mechanical and cellular changes enabling the early-stage expansion of lymph nodes have been characterized, yet the durability of such responses and their implications for adaptive immunity and vaccine efficacy are unknown. Here, by leveraging high-frequency ultrasound imaging of the lymph nodes of mice, we report more potent and persistent lymph-node expansion for animals immunized with a mesoporous silica vaccine incorporating a model antigen than for animals given bolus immunization or standard vaccine formulations such as alum, and that durable and robust lymph-node expansion was associated with vaccine efficacy and adaptive immunity for 100 days post-vaccination in a mouse model of melanoma. Immunization altered the mechanical and extracellular-matrix properties of the lymph nodes, drove antigen-dependent proliferation of immune and stromal cells, and altered the transcriptional features of dendritic cells and inflammatory monocytes. Strategies that robustly maintain lymph-node expansion may result in enhanced vaccination outcomes.
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Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.
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Achilles tendon rupture is a common sports-related tendon injury. Even with advanced clinical treatments, many patients suffer from long-term pain and reduced function. These unsatisfactory outcomes result primarily from an imbalanced injury response with excessive inflammation and inadequate regeneration. Prior studies showed that extracellular vesicles from inflammation-primed adipose-derived stem cells (iEVs) can attenuate inflammation in the early phase of tendon healing. However, the effect of iEVs on tendon inflammation and regeneration in the later phases of tendon healing and the underlying mechanism remain to be determined. Accordingly, this study investigated the mechanistic roles of iEVs in regulating tendon response to injury using a mouse Achilles tendon injury and repair model in vivo and iEV-macrophage and iEV-tendon cell co-culture models in vitro. Results showed that iEVs promoted tendon anti-inflammatory gene expression and reduced mononuclear cell infiltration in the remodeling phase of tendon healing. iEVs also increased injury site collagen deposition and promoted tendon structural recovery. As such, mice treated with iEVs showed less peritendinous scar formation, much lower incidence of postoperative tendon gap or rupture, and faster functional recovery compared to untreated mice. Further in vitro study revealed that iEVs both inhibited macrophage inflammatory response and increased tendon cell proliferation and collagen production. The iEV effects were partially mediated by miR-147-3p, which blocks the toll-like receptor 4/NF-κB signaling pathway that activates macrophage M1 polarization. The combined results demonstrated that iEVs are a promising therapeutic agent, which can enhance tendon repair by attenuating inflammation and promoting intrinsic healing. Significance statement: Using a clinically relevant mouse Achilles tendon injury and repair model, this study revealed that iEVs, a biological product generated from inflammation-primed adipose-derived stem cells, can directly target both macrophages and tendon cells and enhance tendon structural and functional recovery by limiting inflammation and promoting intrinsic healing. Results further identified miR-147-3p as one of the active components of iEVs that modulate macrophage inflammatory response by inhibiting toll-like receptor 4/NF-κB signaling pathway. These promising findings paved the road toward clinical application of iEVs in the treatment of tendon injury and other related disorders.
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Enriched in glycolytic enzymes, paucicellular and hypovascular intrasynovial flexor tendons fail to mount an effective healing response after injury and repair. In contrast, well-vascularized extrasynovial flexor tendons possess high levels of oxidative phosphorylation (OXPHOS) enzymes and have a markedly improved healing capacity. This study was designed to compare the metabolic profiles of the two types of tendons and to evaluate the impact of metabolic reprogramming on early intrasynovial tendon healing in a clinically relevant canine model. Results showed that healthy intrasynovial tendons expressed higher levels of PDK1 and GAPDH and lower levels of SCX and IGF1 than did extrasynovial tendons. PDK1 encodes a subtype of pyruvate dehydrogenase kinase (PDK) that inhibits OXPHOS. Consistently, ATP production via glycolysis was favored in intrasynovial tendon cells whereas OXPHOS was the preferred pathway in extrasynovial tendon cells. Inhibition of glycolysis in vitro increased SCX expression in intrasynovial tendon cells. Therefore, dichloroacetate (DCA), a PDK1 inhibitor, was used in vivo to shift intrasynovial tendon ATP production from glycolysis to OXPHOS. Oral DCA administration reduced serum lactate concentration and increased acetyl-CoA content in repaired intrasynovial tendons and led to reduced TLR4 and IL1B and increased IGF1, SCX, and TGFB3 expressions in treated intrasynovial tendons compared to controls. Immunohistochemistry staining with anti-Ki67 and anti-CD31 antibodies revealed marked increases in cellularity and neovascularization in treated intrasynovial tendons. Clinical significance: The findings of this experiment indicate that improved gene expression and histological outcomes can be achieved by regulating glucose metabolism in the early stages following intrasynovial tendon repair.
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Procedimentos de Cirurgia Plástica , Tendões , Animais , Cães , Trifosfato de Adenosina/metabolismo , Procedimentos de Cirurgia Plástica/veterinária , Tendões/fisiologia , Tendões/cirurgiaRESUMO
The highly variable clinical outcomes noted after intrasynovial tendon repair have been associated with an early inflammatory response leading to the development of fibrovascular adhesions. Prior efforts to broadly suppress this inflammatory response have been largely unsuccessful. Recent studies have shown that selective inhibition of IkappaB kinase beta (IKK-ß), an upstream activator of nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) signaling, mitigates the early inflammatory response and leads to improved tendon healing outcomes. In the current study, we test the hypothesis that oral treatment with the IKK-ß inhibitor ACHP (2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinenitrile an inhibitor) will modulate the postoperative inflammatory response and improve intrasynovial flexor tendon healing. To test this hypothesis, the flexor digitorum profundus tendon of 21 canines was transected and repaired within the intrasynovial region and assessed after 3 and 14 days. Histomorphometry, gene expression analyses, immunohistochemistry, and quantitative polarized light imaging were used to examine ACHP-mediated changes. ACHP led to reduction in phosphorylated p-65, indicating that NF-κB activity was suppressed. ACHP enhanced expression of inflammation-related genes at 3 days and suppressed expression of these genes at 14 days. Histomorphometry revealed enhanced cellular proliferation and neovascularization in ACHP-treated tendons compared with time-matched controls. These findings demonstrate that ACHP effectively suppressed NF-κB signaling and modulated early inflammation, leading to increased cellular proliferation and neovascularization without stimulating the formation of fibrovascular adhesions. Together, these data suggest that ACHP treatment accelerated the inflammatory and proliferative phases of tendon healing following intrasynovial flexor tendon repair. Clinical Significance: Using a clinically relevant large-animal model, this study revealed that targeted inhibition of nuclear factor kappa-light chain enhancer of activated B cells signaling with ACHP provides a new therapeutic strategy for enhancing the repair of sutured intrasynovial tendons.
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NF-kappa B , Tendões , Animais , Cães , Transdução de Sinais , Proteínas Serina-Treonina Quinases , InflamaçãoRESUMO
Cooperativity and antagonism between transcription factors (TFs) can drastically modify their binding to regulatory DNA elements. While mapping these relationships between TFs is important for understanding their context-specific functions, existing approaches either rely on DNA binding motif predictions, interrogate one TF at a time, or study individual TFs in parallel. Here, we introduce paired yeast one-hybrid (pY1H) assays to detect cooperativity and antagonism across hundreds of TF-pairs at DNA regions of interest. We provide evidence that a wide variety of TFs are subject to modulation by other TFs in a DNA region-specific manner. We also demonstrate that TF-TF relationships are often affected by alternative isoform usage and identify cooperativity and antagonism between human TFs and viral proteins from human papillomaviruses, Epstein-Barr virus, and other viruses. Altogether, pY1H assays provide a broadly applicable framework to study how different functional relationships affect protein occupancy at regulatory DNA regions.
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Infecções por Vírus Epstein-Barr , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ligação Proteica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , DNA/metabolismo , Sítios de LigaçãoRESUMO
TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.
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Antígeno B7-H1 , Neoplasias da Mama , Linfócitos T CD8-Positivos , Inibidores de Checkpoint Imunológico , Fator de Crescimento Transformador beta , Feminino , Animais , Camundongos , Diferenciação Celular , Linfócitos T CD8-Positivos/imunologia , Células-Tronco , Antígeno B7-H1/antagonistas & inibidores , Fator de Crescimento Transformador beta/antagonistas & inibidores , Interferon gama/imunologia , Exaustão das Células T , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos Endogâmicos BALB C , Linhagem Celular Tumoral , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , RNA-SeqRESUMO
Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope. The workflow consists of acquiring large field of view fluorescence microscopy (FM) images, which guide to regions of interest for successive EM (integrated correlative light and electron microscopy). High precision EM-FM overlay is achieved using cathodoluminescent markers. We conduct a proof-of-concept of our integrated workflow on immunolabelled serial sections of tissues. Acquisitions are limited to regions containing biological targets, expediting total acquisition times and reducing the burden of excess data by tens or hundreds of GBs.
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Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 h. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy.
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The success of immunotherapy with immune checkpoint inhibitors (ICIs) in a subset of individuals has been very exciting. However, in many cancers, responses to current ICIs are modest and are seen only in a small subsets of patients. Herein, a widely applicable approach that increases the benefit of ICIs is reported. Intratumoral administration of augmenting immune response and inhibiting suppressive environment of tumors-AIRISE-02 nanotherapeutic that co-delivers CpG and STAT3 siRNA-results in not only regression of the injected tumor, but also tumors at distant sites in multiple tumor model systems. In particular, three doses of AIRISE-02 in combination with systemic ICIs completely cure both treated and untreated aggressive melanoma tumors in 63% of mice, while ICIs alone do not cure any mice. A long-term memory immune effect is also reported. AIRISE-02 is effective in breast and colon tumor models as well. Lastly, AIRISE-02 is well tolerated in mice and nonhuman primates. This approach combines multiple therapeutic agents into a single nanoconstruct to create whole-body immune responses across multiple cancer types. Being a local therapeutic, AIRISE-02 circumvents regulatory challenges of systemic nanoparticle delivery, facilitating rapid translation to the clinic. AIRISE-02 is under investigational new drug (IND)-enabling studies, and clinical trials will soon follow.
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Imunoterapia , Nanopartículas , RNA Interferente Pequeno , Animais , Camundongos , VacinaçãoRESUMO
Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.