Identification of hypoxic macrophages in glioblastoma with therapeutic potential for vasculature normalization.

Monocyte-derived tumor-associated macrophages (Mo-TAMs) intensively infiltrate diffuse gliomas with remarkable heterogeneity. Using single-cell transcriptomics, we chart a spatially resolved transcriptional landscape of Mo-TAMs across 51 patients with isocitrate dehydrogenase (IDH)-wild-type glioblastomas or IDH-mutant gliomas. We characterize a Mo-TAM subset that is localized to the peri-necrotic niche and skewed by hypoxic niche cues to acquire a hypoxia response signature. Hypoxia-TAM destabilizes endothelial adherens junctions by activating adrenomedullin paracrine signaling, thereby stimulating a hyperpermeable neovasculature that hampers drug delivery in glioblastoma xenografts. Accordingly, genetic ablation or pharmacological blockade of adrenomedullin produced by Hypoxia-TAM restores vascular integrity, improves intratumoral concentration of the anti-tumor agent dabrafenib, and achieves combinatorial therapeutic benefits. Increased proportion of Hypoxia-TAM or adrenomedullin expression is predictive of tumor vessel hyperpermeability and a worse prognosis of glioblastoma. Our findings highlight Mo-TAM diversity and spatial niche-steered Mo-TAM reprogramming in diffuse gliomas and indicate potential therapeutics targeting Hypoxia-TAM to normalize tumor vasculature.

Boosted photo-immunotherapy via near-infrared light excited phototherapy in tumor sites and photo-activation in sentinel lymph nodes.

Phototherapy is a promising modality that could eradicate tumor and trigger immune responses via immunogenic cell death (ICD) to enhance anti-tumor immunity. However, due to the lack of deep-tissue-excitable phototherapeutic agents and appropriate excitation strategies, the utility of phototherapy for efficient activation of the immune system is challenging. Herein, we report functionalized ICG nanoparticles (NPs) with the capture capability of tumor-associated antigens (TAAs). Under near-infrared (NIR) light excitation, the ICG NPs exhibited high-performance phototherapy, i.e., synergistic photothermal therapy and photodynamic therapy, thereby efficiently eradicating primary solid tumor and inducing ICD and subsequently releasing TAAs. The ICG NPs also captured TAAs and delivered them to sentinel lymph nodes, and then the sentinel lymph nodes were activated with NIR light to trigger efficient T-cell immune responses through activation of dendritic cells with the assistance of ICG NP generated reactive oxygen species, inhibiting residual primary tumor recurrence and controlling distant tumor growth. The strategy of NIR light excited phototherapy in tumor sites and photo-activation in sentinel lymph nodes provides a powerful platform for active immune systems for anti-tumor photo-immunotherapy.

Intravital imaging of splenic classical monocytes modifying the hepatic CX3CR1+ cells motility to exacerbate liver fibrosis via spleen-liver axis.

CX3CR1+ cells play a crucial role in liver fibrosis progression. However, changes in the migratory behavior and spatial distribution of spleen-derived and hepatic CX3CR1+ cells in the fibrotic liver as well as their influence on the liver fibrosis remain unclear. METHODS: The CX3CR1GFP/+ transgenic mice and CX3CR1-KikGR transgenic mice were used to establish the CCl4-induced liver fibrosis model. Splenectomy, adoptive transfusion of splenocytes, in vivo photoconversion of splenic CX3CR1+ cells and intravital imaging were performed to study the spatial distribution, migration and movement behavior, and regulatory function of CX3CR1+ cells in liver fibrosis. RESULTS: Intravital imaging revealed that the CX3CR1GFP cells accumulated into the fibrotic liver and tended to accumulate towards the central vein (CV) in the hepatic lobules. Two subtypes of hepatic CX3CR1+ cells existed in the fibrotic liver. The first subtype was the interacting CX3CR1GFP cells, most of which were observed to distribute in the liver parenchyma and had a higher process velocity; the second subtype was mobile CX3CR1GFP cells, most of which were present in the hepatic vessels with a faster moving speed. Splenectomy ameliorated liver fibrosis and decreased the number of CX3CR1+ cells in the fibrotic liver. Moreover, splenectomy rearranged CX3CR1GFP cells to the boundary of the hepatic lobule, reduced the process velocity of interacting CX3CR1GFP cells and decreased the number and mobility of mobile CX3CR1GFP cells in the fibrotic liver. Transfusion of spleen-derived classical monocytes increased the process velocity and mobility of hepatic endogenous CX3CR1GFP cells and facilitated liver fibrosis progression via the production of proinflammatory and profibrotic cytokines. The photoconverted splenic CX3CR1+ KikRed+ cells were observed to leave the spleen, accumulate into the fibrotic liver and contact with hepatic CX3CR1+ KikGreen+ cells during hepatic fibrosis. CONCLUSION: The splenic CX3CR1+ monocytes with classical phenotype migrated from the spleen to the fibrotic liver, modifying the migratory behavior of hepatic endogenous CX3CR1GFP cells and exacerbating liver fibrosis via the secretion of cytokines. This study reveals that splenic CX3CR1+ classical monocytes are a key driver of liver fibrosis via the spleen-liver axis and may be potential candidate targets for the treatment of chronic liver fibrosis.

Intravital imaging of the functions of immune cells in the tumor microenvironment during immunotherapy.

Cancer immunotherapy has developed rapidly in recent years and stands as one of the most promising techniques for combating cancer. To develop and optimize cancer immunotherapy, it is crucial to comprehend the interactions between immune cells and tumor cells in the tumor microenvironment (TME). The TME is complex, with the distribution and function of immune cells undergoing dynamic changes. There are several research techniques to study the TME, and intravital imaging emerges as a powerful tool for capturing the spatiotemporal dynamics, especially the movement behavior and the immune function of various immune cells in real physiological state. Intravital imaging has several advantages, such as high spatio-temporal resolution, multicolor, dynamic and 4D detection, making it an invaluable tool for visualizing the dynamic processes in the TME. This review summarizes the workflow for intravital imaging technology, multi-color labeling methods, optical imaging windows, methods of imaging data analysis and the latest research in visualizing the spatio-temporal dynamics and function of immune cells in the TME. It is essential to investigate the role played by immune cells in the tumor immune response through intravital imaging. The review deepens our understanding of the unique contribution of intravital imaging to improve the efficiency of cancer immunotherapy.

Demand-driven active droplet generation and sorting based on positive pressure-controlled fluid wall.

Droplet microfluidics is a rapidly advancing area of microfluidic technology, which offers numerous advantages for cell analysis, such as isolation and accumulation of signals, by confining cells within droplets. However, controlling cell numbers in droplets is challenging due to the uncertainty of random encapsulation which result in many empty droplets. Therefore, more precise control techniques are needed to achieve efficient encapsulation of cells within droplets. Here, an innovative microfluidic droplet manipulation platform had been developed, which employed positive pressure as a stable and controllable driving force for manipulating fluid within chips. The air cylinder, electro-pneumatics proportional valve, and the microfluidic chip were connected through a capillary, which enabled the formation of a fluid wall by creating a difference in hydrodynamic resistance between two fluid streams at the channel junction. Lowering the pressure of the driving oil phase eliminates hydrodynamic resistance and breaks the fluid wall. Regulating the duration of the fluid wall breakage controls the volume of the introduced fluid. Several important droplet microfluidic manipulations were demonstrated on this microfluidic platform, such as sorting of cells/droplets, sorting of droplets co-encapsulated cells and hydrogels, and active generation of droplets encapsulated with cells in a responsive manner. The simple, on-demand microfluidic platform was featured with high stability, good controllability, and compatibility with other droplet microfluidic technologies.

Intravital molecular imaging reveals that ROS-caspase-3-GSDME-induced cell punching enhances humoral immunotherapy targeting intracellular tumor antigens.

Tumor antigens (TAs)-induced humoral immune responses or TAs-specific antibodies have great application prospects for tumor therapy. However, more than half of TAs are intracellular antigens (intra-Ags) that are hardly recognized by antibodies. It is worthy to develop immunotherapeutic strategies for targeting intra-Ags. Methods: We used the far-red fluorescent protein tfRFP as an intracellular antigen to immunize mice and generated a liver metastasis model by injecting tfRFP-expressing B16 melanoma cells (tfRFP-B16) via the spleen. Intravital molecular imaging and atomic force microscopy were performed to visualize the formation of tfRFP antigen-antibody complexes (also known as immune complexes) and punched holes in cell membranes. Results: The results showed that the tfRFP-elicited immune responses inhibited the metastasis of tfRFP-expressing melanoma cells in the liver. In the circulating tfRFP-B16 tumor cells, elevated reactive oxygen species (ROS) induced slight caspase-3 activation, a probable key factor in the cleavage of gasdermin E (GSDME) proteins and punching of holes in the tumor cell membrane. Increased tumor cell membrane permeability led to the release of intra-Ag tfRFP and binding with anti-tfRFP antibodies. The formation of tfRFP antigen-antibody complexes on the membranes of tfRFP-B16 cells activated complement components to form membrane attack complexes to further destroy the cell membrane. Neutrophils were rapidly recruited, and F4/80+ macrophages phagocytized the dying tumor cells. Conclusion: The process of circulating tumor cell elimination in the tfRFP-immunized mice was triggered through the ROS-caspase-3-GSDME pathway to form intra-Ag-antibody immune complexes, which were involved in the activation of the complement system, as well as the recruitment of neutrophils and F4/80+ macrophages. An intra-Ag-elicited humoral immune response is a potent strategy for eliminating liver metastasis, which is unaffected by the liver immune tolerogenic status.

Noninvasive and early diagnosis of acquired brain injury using fluorescence imaging in the NIR-II window.

Acquired brain injury (ABI), which is the umbrella term for all brain injuries, is one of the most dangerous diseases resulting in high morbidity and mortality, making it extremely significant to early diagnosis of ABI. Current methods, which are mainly composed of X-ray computed tomography and magnetic resonance angiography, remain limited in diagnosis of ABI with respect to limited spatial resolution and long scanning times. Here, we reported through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy, utilizing the fluorescence of down-conversion nanoparticles (DCNPs) in the 1.3 - 1.7 µm near-infrared window (NIR-II window). Due to its high spatial resolution of 22.79 µm, the NIR-II fluorescence imaging method could quickly distinguish the brain injury region of mice after performing the stab wound injury (traumatic brain injury) and ischemic stroke (non-traumatic brain injury), enabling it a powerful tool in the noninvasive and early diagnosis of ABI.

Near-infrared light excited photodynamic anticancer therapy based on UCNP@AIEgen nanocomposite.

Photodynamic therapy (PDT), a clinically approved cancer treatment strategy, features non-invasiveness, few side-effects, high spatial resolution, etc. The advancement of PDT has been significantly restricted by the penetration depth of the excitation light. Herein, an effective fluorogen, TBD, with aggregation-induced emission characteristics (AIEgen) and high reactive-oxygen-species (ROS) generation efficiency was reported and integrated with a near infrared (NIR) light excitable upconversion nanoparticle (UCNP) to construct NIR light excitable UCNP@TBD nanocomposites. The formed nanocomposite has excellent photostability, good biocompatibility, and efficient ROS generation under NIR light excitation via Förster resonance energy transfer (FRET), enabling NIR light excited PDT. Moreover, the proposed NIR light excited PDT can break the impasse between the penetration depth and excitation volume in conventional PDT, effectively improving the anticancer therapeutic outcome. In vitro cancer cell ablation and in vivo tumor growth inhibition validated that the proposed UCNP@TBD nanocomposite is a promising NIR light excitable PDT agent with great potential for future translational research.

Corrigendum to "I-alowCD11bhigh DC Regulates the Immune Response in the Eyes of Experimental Autoimmune Uveitis".

[This corrects the article DOI: 10.1155/2020/6947482.].

Targeted immunomodulation of inflammatory monocytes across the blood-brain barrier by curcumin-loaded nanoparticles delays the progression of experimental autoimmune encephalomyelitis.

It is difficult to carry out early diagnosis and treatment of Multiple sclerosis (MS) because of the complex pathogenesis elicited by diversified autoantigens. Monocytes play important roles in the process of MS, especially as most of the amplified inflammatory monocytes cross the BBB to promote neuron injury and recruit more immune cells to infiltrate the central nervous system (CNS). Here, we propose monocytes as an effective immunotherapy target for MS. We used High-density lipoprotein-mimicking peptide-phospholipid scaffold (HPPS) as a carrier to improve the bioavailability of curcumin. Curcumin-loaded HPPS (Cur-HPPS) were taken up specifically and efficiently by monocytes through the scavenger receptor class B type I (SR-B1) receptor. This delivery hindered inflammatory monocytes across the BBB in EAE mice, inhibited the proliferation of microglia, and restricted the infiltration of other effector immune cells, resulting in the reduction of EAE morbidity from 100% to 30%. It attributed to the immunomodulatory effect of Cur-HPPS on inflammatory monocytes, which inhibited NF-κB activation and downregulated the expression of adhesion-and migration-related molecules. Meanwhile, infiltrated monocytes in the CNS of EAE mice characterize early inflammation. Therefore, targeted modulation of monocytes with HPPS carrying therapeutic and/or imaging agents offers a novel strategy for MS diagnosis and treatment.

I-alowCD11bhigh DC Regulates the Immune Response in the Eyes of Experimental Autoimmune Uveitis.

Regulatory dendritic cells (DCreg) have been reported to be a negative regulator in the immune response. These cells are widely distributed in the liver, spleen, and lung. However, the status and function of DCreg in the eyes and disease are still not very clear. Herein, we found that the number of I-alowCD11bhigh DC increased in the eye and spleen at the recovery stage of experimental autoimmune uveitis (EAU), which is a mouse model for autoimmune uveitis. These cells expressed lower levels of CD80, CD86, and CD54 than the mature DCs and expressed interleukin 10 (IL-10), indoleamine 2,3-dioxygenase (IDO), and transforming growth factor beta (TGF-ß) as well. Moreover, these DCreg can regulate the development of EAU by promoting CD4+CD25+Foxp3+ regulatory T cells. The increased interferon-gamma (IFN-γ) in the aqueous humor of EAU participates in inducing DCreg to alleviate the symptom of EAU. Furthermore, DCreg was found to exist in the eyes of normal mice. Aqueous humor, containing a certain concentration of IL-10, TGF-ß, prostaglandin E2 (PGE2), IDO, and nitric oxide (NO), induced the tolerance of DCreg in normal eyes. It can be concluded that DCreg exists in the eyes and plays a protective role in inflamed eyes. These DCreg induced by IFN-γ might be used as a strategy to develop therapy for EAU management.

Melittin-lipid nanoparticles target to lymph nodes and elicit a systemic anti-tumor immune response.

Targeted delivery of a nanovaccine loaded with a tumor antigen and adjuvant to the lymph nodes (LNs) is an attractive approach for improving cancer immunotherapy outcomes. However, the application of this technique is restricted by the paucity of suitable tumor-associated antigens (TAAs) and the sophisticated technology required to identify tumor neoantigens. Here, we demonstrate that a self-assembling melittin-lipid nanoparticle (α-melittin-NP) that is not loaded with extra tumor antigens promotes whole tumor antigen release in situ and results in the activation of antigen-presenting cells (APCs) in LNs. Compared with free melittin, α-melittin-NPs markedly enhance LN accumulation and activation of APCs, leading to a 3.6-fold increase in antigen-specific CD8+ T cell responses. Furthermore, in a bilateral flank B16F10 tumor model, primary and distant tumor growth are significantly inhibited by α-melittin-NPs, with an inhibition rate of 95% and 92%, respectively. Thus, α-melittin-NPs induce a systemic anti-tumor response serving as an effective LN-targeted whole-cell nanovaccine.

Neutrophil infiltration and whole-cell vaccine elicited by N-dihydrogalactochitosan combined with NIR phototherapy to enhance antitumor immune response and T cell immune memory.

Melanoma is one of the deadliest malignancies with a high risk of relapse and metastasis. Long-term, tumor-specific, and systemic immunity induced by local intervention is ideal for personalized cancer therapy. Laser immunotherapy (LIT), a combination of local irradiation of laser and local administration of an immunostimulant, was developed to achieve such an immunity. Although LIT showed promising efficacy on tumors, its immunological mechanism is still not understood, especially its spatio-temporal dynamics. Methods: In this study, we investigated LIT-induced immunological responses using a 980-nm laser and a novel immunostimulant, N-dihydrogalactochitosan (GC). Then we followed the functions of key immune cells spatially and temporally using intravital imaging and immunological assays. Results: Immediately after LIT, GC induced a rapid infiltration of neutrophils which ingested most GC in tumors. The cytokines released to the serum peaked at 12 h after LIT. Laser irradiations produced photothermal effects to ablate the tumor, release damage-associated molecular patterns, and generate whole-cell tumor vaccines. LIT-treated tumor-bearing mice efficiently resisted the rechallenged tumor and prevented lung metastasis. Intravital imaging of tumor at rechallenging sites in LIT-treated mice revealed that the infiltration of tumor-infiltrating lymphocytes (TILs) increased with highly active motility. Half of TILs with arrest and confined movements indicated that they had long-time interactions with tumor cells. Furthermore, LIT has synergistic effect with checkpoint blockade to improve antitumor efficacy. Conclusion: Our research revealed the important role of LIT-induced neutrophil infiltration on the in situ whole-cell vaccine-elicited antitumor immune response and long-term T cell immune memory.

Dynamic visualization of the whole process of cytotoxic T lymphocytes killing B16 tumor cells in vitro.

Cytotoxic T lymphocytes (CTLs) play a key role in adoptive cell therapy (ACT) by destroying tumor cells. Although some mechanisms of CTLs killing tumor cells have already been revealed, the precise dynamic information of CTLs' interaction with tumor cells is still not known. Here, we used confocal microscopy to visualize the whole process of how CTLs kill tumor cells in vitro. According to imaging data, CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or more tumor cells, and the average time for CTLs destroying one or more tumor cells in vitro is dozens of minutes only. Our study displayed the temporal events of CTLs' interaction with tumor cells at the beginning up to the point of killing them. Furthermore, the imaging data presented strong cytotoxicity of CTLs toward the specific tumor cells. These results could help us to well understand the mechanism of CTLs' elimination of tumor cells and improve the efficacy of ACT in cancer immunotherapy.

Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy.

The combined-immunotherapy of adoptive cell therapy (ACT) and cyclophosphamide (CTX) is one of the most efficient treatments for melanoma patients. However, no synergistic effects of CTX and ACT on the spatio-temporal dynamics of immunocytes in vivo have been described. Here, we visualized key cell events in immunotherapy-elicited immunoreactions in a multicolor-coded tumor microenvironment, and then established an optimal strategy of metronomic combined-immunotherapy to enhance anti-tumor efficacy. Intravital imaging data indicated that regulatory T cells formed an 'immunosuppressive ring' around a solid tumor. The CTX-ACT combined-treatment elicited synergistic immunoreactions in tumor areas, which included relieving the immune suppression, triggering the transient activation of endogenous tumor-infiltrating immunocytes, increasing the accumulation of adoptive cytotoxic T lymphocytes, and accelerating the infiltration of dendritic cells. These insights into the spatio-temporal dynamics of immunocytes are beneficial for optimizing immunotherapy and provide new approaches for elucidating the mechanisms underlying the involvement of immunocytes in cancer immunotherapy.

In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity.

In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP(+) microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo.

Zigzag Generalized Lévy Walk: the In Vivo Search Strategy of Immunocytes.

Immune responses are based on the coordinated searching behaviors of immunocytes that are aimed at tracking down specific targets. The search efficiency of immunocytes significantly affects the speed of initiation and development of immune responses. Previous studies have shown that not only the intermittent walk but also the zigzag turning preference of immunocytes contributes to the search efficiency. However, among existing models describing immunocytes' search strategy, none has captured both features. Here we propose a zigzag generalized Lévy walk model to describe the search strategy of immunocytes more accurately and comprehensively by considering both the intermittent and the zigzag-turning walk features. Based on the analysis of the searching behaviors of typical immune cell types, dendritic cells and leukocytes, in their native physiological environment, we demonstrate that the model can describe the in vivo search strategy of immunocytes well. Furthermore, by analyzing the search efficiency, we find that this type of search strategy enables immunocytes to capture rare targets in approximately half the time than the previously proposed generalized Lévy walk. This study sheds new light on the fundamental mechanisms that drive the efficient initiation and development of immune responses and in turn may lead to the development of novel therapeutic approaches for diseases ranging from infection to cancer.

Hybrid melittin cytolytic Peptide-driven ultrasmall lipid nanoparticles block melanoma growth in vivo.

The cytolytic peptide melittin is a potential anticancer candidate that may be able to overcome tumor drug resistance due to its lytic properties. However, in vivo applications of melittin are limited due to its main side effect, hemolysis, which is especially pronounced following intravenous administration. Here, we designed a hybrid cytolytic peptide, α-melittin, in which the N-terminus of melittin is linked to the C-terminus of an amphipathic α-helical peptide (α-peptide) via a GSG linker. The strong α-helical configuration allows α-melittin to interact with phospholipids and self-assemble into lipid nanoparticles, with a high efficiency for α-melittin encapsulation (>80%) and a strong ability to control the structure of the nanoparticle (~20 nm). This α-melittin-based lipid nanoparticle (α-melittin-NP) efficiently shields the positive charge of melittin (18.70 ± 0.90 mV) within the phospholipid monolayer, resulting in the generation of a neutral nanoparticle (2.45 ± 0.56 mV) with reduced cytotoxicity and a widened safe dosage range. Confocal imaging data confirmed that α-melittin peptides were efficiently released from the nanoparticles and were cytotoxic to the melanoma cells. Finally, α-melittin-NPs were administered to melanoma-bearing mice via intravenous injection. The growth of the melanoma cells was blocked by the α-melittin-NPs, with an 82.8% inhibition rate relative to the PBS-treated control group. No side effects of treatment were found in this study. Thus, the excellent properties of α-melittin-NP give it potential clinical applications in solid tumor therapeutics through intravenous administration.

Monitoring the process of pulmonary melanoma metastasis using large area and label-free nonlinear optical microscopy.

We performed large area nonlinear optical microscopy (NOM) for label-free monitoring of the process of pulmonary melanoma metastasis ex vivo with subcellular resolution in C57BL/6 mice. Multiphoton autofluorescence (MAF) and second harmonic generation (SHG) images of lung tissue are obtained in a volume of ≈ 2.2 mm × 2.2 mm × 30 µm. Qualitative differences in morphologic features and quantitative measurement of pathological lung tissues at different time points are characterized. We find that combined with morphological features, the quantitative parameters, such as the intensity ratio of MAF and SHG between pathological tissue and normal tissue and the MAF to SHG index versus depth clearly shows the tissue physiological changes during the process of pulmonary melanoma metastasis. Our results demonstrate that large area NOM succeeds in monitoring the process of pulmonary melanoma metastasis, which can provide a powerful tool for the research in tumor pathophysiology and therapy evaluation.