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Liver sinusoidal endothelial cells (LSECs) are responsible for the immunologic tolerance of liver which is a common site for visceral metastases, suggesting its potential role as an target for cancer immunotherapy. However, targeted modulation of LSECs is still not achieved thus far. Here, we report LSECs are specifically targeted and modulated by melittin nanoparticles (α-melittin-NPs). Intravital imaging shows that LSECs fluoresce within 20 s after intravenous injection of α-melittin-NPs. α-melittin-NPs trigger the activation of LSECs and lead to dramatic changes of cytokine/chemokine milieu in the liver, which switches the hepatic immunologic environment to the activated state. As a result, α-melittin-NPs resist the formation of metastatic lesions with high efficiency. More strikingly, the survival rate reaches 80% in the spontaneous liver metastatic tumor model. Our research provides support for the use of α-melittin-NPs to break LSEC-mediated immunologic tolerance, which opens an avenue to control liver metastasis through the immunomodulation of LSECs. Liver sinusoidal endothelial cells are known to promote immune tolerance in liver. Here, the authors target these cells using melittin nanoparticles and show alterations in the liver immune environment and suppression of liver metastases.

Cytotoxic T lymphocytes (CTLs) and their gene-engineered cells display great application prospects in tumor immunotherapy. The timing of CTL-induced molecular events in tumor cells is unclear, and we also unknow whether the killing efficiency of CTLs is restrained in the liver, an immunotolerant organ with a high tumor incidence. We used intravital imaging to dynamically monitor the fluorescence resonance energy transfer (FRET) signals of caspase-3 and calcium sensor in tumor cells after transferring CTLs into tumor-bearing mice. Our data show that several CTLs attacked on one tumor cell, and on average each CTL killed 1.24 ± 0.11 tumor cells per day in the liver, which was much less efficient than that in the spleen (3.18 ± 0.26 tumor cells/CTL/day). The killing efficiency of CTLs is restrained in the liver and can be reversed by blocking immunosuppressive cytokine. Tumor cells exposed to CTLs appeared to have prolonged calcium influx, which occurred dozens of minutes before caspase-3 activity. The quantitative characterization of these molecular and cellular events provides accurate information to evaluate the efficiency of cellular immunotherapy against tumors and understand the impact of an organ's immune status.

Ferroptosis, a recently elucidated style of regulated cell death, has emerged as a significant area of investigation in cancer biology. Natural active compounds that have anti-cancer effects are promising candidates for cancer prevention. Iberverin, a natural compound derived from Brassica oleracea var. capitata, has been shown to exert anti-tumor activities in some cancers. However, its role in hepatocellular carcinoma (HCC) cells and the molecular mechanisms are still poorly understood. In this study, we proved that iberverin can induce intracellular reactive oxygen species (ROS) generation to inhibit cell proliferation and initiate ferroptotic cell death in HCC cells, which can be eradicated by the ferroptosis inhibitor ferrostatin-1 (Fer-1) or deferoxamine mesylate (DFO) and ROS scavenger (GSH or NAC). Mechanistically, iberverin treatment can simultaneously downregulate SLC7A11 mRNA level and degrade GPX4 through the ubiquitination pathway, leading to lipid peroxidation and ferroptotic cell death in HCC cells. Significantly, a low dose of iberverin can remarkably increase the sensitivity of HCC cells to ferroptosis induced by canonical ferroptosis inducers RSL3 and imidazole ketone erastin (IKE). This study uncovers a critical function of iberverin in preventing HCC through ferroptosis and provides a promising strategy for HCC treatment either via iberverin alone or in combination with canonical ferroptosis inducers in the future.

The use of optical microscopy and labeling methods in intravital imaging allows for direct tracking of cell behavior and dynamic changes at the molecular level in the physiological or pathological microenvironment of living animals, revealing the spatiotemporal information of individual cells in the immune response. The liver is an immunological organ that contains unique innate and adaptive immune cells, including Kupffer cells (KCs) and different types of T cells, and is involved in coordinating multiple immune responses in the body. Using intravital imaging to visualize the movement behaviors and functions of immune cells during the reaction processes of the liver under physiological and pathological conditions has shed new light on the understanding of liver immunity, which is of great significance for the diagnosis and treatment of liver diseases. This review introduces various window models and labeling methods for the liver in intravital optical imaging and describes how it provides movement behavior and functional information about different types of immune cells, such as KCs and T cells, in the liver. Additionally, we highlight recent advances in intravital optical imaging of liver diseases, such as nonalcoholic fatty liver disease, infections, and tumors. This review aims to be a useful resource for comprehending the developments and achievements in intravital imaging of the liver and uncovering spatiotemporal information of immune response in a living microenvironment. This review introduces intravital imaging of the liver, including microscopic optical imaging technologies, window models and optical labeling methods, describing how intravital imaging provides movement behavior and functional information of different immune cells in the liver and exhibiting recent advances in intravital optical imaging of liver diseases. We believe this review can be a useful resource for comprehending and uncovering the dynamic process and spatiotemporal information of immune response in a living microenvironment.

The natural product auraptene can influence tumor cell proliferation and invasion, but its effect on hepatocellular carcinoma (HCC) cells is unknown. Here, we report that auraptene can exert anti-tumor effects in HCC cells via inhibition of cell proliferation and ferroptosis induction. Auraptene treatment induces total ROS and lipid ROS production in HCC cells to initiate ferroptosis. The cell death or cell growth inhibition of HCC cells induced by auraptene can be eliminated by the ROS scavenger NAC or GSH and ferroptosis inhibitor ferrostatin-1 or Deferoxamine Mesylate (DFO). Mechanistically, the key ferroptosis defense protein SLC7A11 is targeted for ubiquitin–proteasomal degradation by auraptene, resulting in ferroptosis of HCC cells. Importantly, low doses of auraptene can sensitize HCC cells to ferroptosis induced by RSL3 and cystine deprivation. These findings demonstrate a critical mechanism by which auraptene exhibits anti-HCC effects via ferroptosis induction and provides a possible therapeutic strategy for HCC by using auraptene or in combination with other ferroptosis inducers.

The liver has a unique vascular structure and regional immunosuppressive characteristics closely linked to the occurrence and development of diseases. There are no long-term, large-field, and high-quality imaging methods to simultaneously obtain the structure of blood vessels and movement activities of immune cells in abdominal organs This research developed a drawer-type abdominal window with an acrylic/resin coverslip named DAWarc and applied it to the intravital fluorescence/photoacoustic imaging of the liver for over 10 days. The liver lobe was inserted into the drawer holder of the DAWarc to physically fix the liver, which decreased the imaging artifacts. The acrylic/resin material used as the coverslip has a high compatibility for fluorescence/photoacoustic imaging. Through intravital fluorescence/photoacoustic imaging, information on the structure of hepatic lobules, spatial distribution of nanopomegranate labeled Kupffer cells (KCs), the movement behavior of invariant natural killer T cells, and morphology of KCs were obtained. We also used a self-organizing map neural network to detect tumor metastases in the photoacoustic images automatically. Conclusively, the DAWarc model provided a powerful tool for intravital fluorescence/photoacoustic imaging of the liver; it helped us to better understand the structure of hepatic lobules and the distribution and function of immune cells during the occurrence and development of liver diseases.

The crosstalk between tumor progression and ferroptosis is largely unknown. Here, we identify malate dehydrogenase 2 (MDH2) as a key regulator of ferroptosis. MDH2 deficiency inhibits the growth of hepatocellular carcinoma (HCC) cells and enhances their sensitivity to ferroptosis induced by RAS-selective lethal 3 (RSL3), a compound known to cause ferroptosis. MDH2 knock-down enhances RSL3-induced intracellular reactive oxygen species, free iron ions and lipid per-oxides levels, leading to HCC ferroptotic cell death which is rescued by ferrostatin-1 and iron chelator deferiprone. Importantly, the inhibition of HCC cell growth caused by MDH2 deficiency is partially rescued by ferroptosis blockade. Mechanistically, MDH2 resists RSL3-induced ferroptosis sensitivity dependent on glutathione peroxidase 4 (GPX4), an enzyme responsible for scavenging lipid peroxides, which is stabilized by MDH2 in HCC. The protein expressions of MDH2 and GPX4 are positively correlated with each other in HCC cell lines. Furthermore, through our UALCAN website analysis, we found that MDH2 and GPX4 are highly expressed in HCC samples. These findings reveal a critical mechanism by which HCC evades ferroptosis via MDH2-mediated stabilization of GPX4 to promote tumor progression and underscore the potential of MDH2 inhibition in combi-nation with ferroptosis inducers for the treatment of HCC.

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. The CX3CR1 transgenic mice and CX3CR1-KikGR transgenic mice were used to establish the CCl4-induced liver fibrosis model. Splenectomy, adoptive transfusion of splenocytes, 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. Intravital imaging revealed that the CX3CR1 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 CX3CR1 cells, most of which were observed to distribute in the liver parenchyma and had a higher process velocity; the second subtype was mobile CX3CR1 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 CX3CR1 cells to the boundary of the hepatic lobule, reduced the process velocity of interacting CX3CR1 cells and decreased the number and mobility of mobile CX3CR1 cells in the fibrotic liver. Transfusion of spleen-derived classical monocytes increased the process velocity and mobility of hepatic endogenous CX3CR1 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. The splenic CX3CR1 monocytes with classical phenotype migrated from the spleen to the fibrotic liver, modifying the migratory behavior of hepatic endogenous CX3CR1 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.

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. 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) 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. 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. 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.

The role of neutrophils in bone regeneration remains elusive. In this study, it is shown that intramuscular implantation of interleukin‐8 (IL‐8) (commonly recognized as a chemotactic cytokine for neutrophils) at different levels lead to outcomes resembling those of fracture hematoma at various stages. Ectopic endochondral ossification is induced by certain levels of IL‐8, during which neutrophils are recruited to the implanted site and are N2‐polarized, which then secrete stromal cell‐derived factor‐1α (SDF‐1α) for bone mesenchymal stem cell (BMSC) chemotaxis via the SDF‐1/CXCR4 (C‐X‐C motif chemokine receptor 4) axis and its downstream phosphatidylinositol 3'‐kinase (PI3K)/Akt pathway and β‐catenin‐mediated migration. Neutrophils are pivotal for recruiting and orchestrating innate and adaptive immunocytes, as well as BMSCs at the initial stage of bone healing and regeneration. The results in this study delineate the mechanism of neutrophil‐initiated bone regeneration and interaction between neutrophils and BMSCs, and innate and adaptive immunities. This work lays the foundation for research in the fields of bone regenerative therapy and biomaterial development, and might inspire further research into novel therapeutic options. Following inflammatory stimuli at the bone defect site, neutrophils arrive first, recruit and orchestrate innate and adaptive immunocytes and BMSCs to initiate endochondral ossification under the appropriate immune environment. In this process, N2‐polarized neutrophils mediate anti‐inflammatory transformation of macrophages and CD4+ T cells, secrete SDF‐1α for BMSC chemotaxis via the SDF‐1/CXCR4 axis and its downstream PI3K/AKT pathway and β‐catenin‐mediated migration.