NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

Harnessing Oncolytic Extracellular Vesicles for Tumor Cell-Preferential Cytoplasmic Delivery of Misfolded Proteins for Cancer Immunotherapy.

In this study, extracellular vesicles (EVs) are reimagined as more than just a cellular waste disposal system and are repurposed for cancer immunotherapy. Potent oncolytic EVs (bRSVF-EVs) loaded with misfolded proteins (MPs) are engineered, which are typically considered cellular debris. By impairing lysosomal function using bafilomycin A1 and expressing the respiratory syncytial virus F protein, a viral fusogen, MPs are successfully loaded into the EVs expressing RSVF. bRSVF-EVs preferentially transplant a xenogeneic antigen onto cancer cell membranes in a nucleolin-dependent manner, triggering an innate immune response. Furthermore, bRSVF-EV-mediated direct delivery of MPs into the cancer cell cytoplasm initiates endoplasmic reticulum stress and immunogenic cell death (ICD). This mechanism of action leads to substantial antitumor immune responses in murine tumor models. Importantly, when combined with PD-1 blockade, bRSVF-EV treatment elicits robust antitumor immunity, resulting in prolonged survival and complete remission in some cases. Overall, the findings demonstrate that utilizing tumor-targeting oncolytic EVs for direct cytoplasmic delivery of MPs to induce ICD in cancer cells represents a promising approach for enhancing durable antitumor immunity.

DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging.

Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called 'DNA skybridge'. The DNA skybridge is constructed in a 3D structure with 4 µm-high thin quartz barriers in a quartz slide. Each DNA end is attached to the top of the adjacent barrier, resulting in the extension and immobilization of DNA. In this 3D structure, the bottom surface is out-of-focus when the target molecules on the DNA are imaged. Moreover, the DNA skybridge itself creates a thin Gaussian light sheet beam parallel to the immobilized DNA. This dual property allows for imaging a single probe-tagged molecule moving on DNA while effectively suppressing interference with the surface and background signals from the surface.

Single-Molecule Co-Immunoprecipitation Reveals Functional Inheritance of EGFRs in Extracellular Vesicles.

Cancer cells actively release extracellular vesicles (EVs) as important carriers of cellular information to tumor microenvironments. Although the composition and quantity of the proteins contained in EVs are characterized, it remains unknown how these proteins in EVs are related to those in the original cells at the functional level. With epidermal growth factor receptor (EGFR) in lung adenocarcinoma cells as a model oncoprotein, it is studied how distinct types of EVs, microvesicles and exosomes, represent their original cells at the protein and protein-protein interaction (PPI) level. Using the recently developed single-molecule immunolabeling and co-immunoprecipitation schemes, the quantity and PPI strengths of EGFRs derived from EVs and the original lung adenocarcinoma cells are determined. It is found that the microvesicles exhibit higher correlations with the original cells than the exosomes in terms of the EGFR levels and their PPI patterns. In spite of these detailed differences between the microvesicles and exosomes, the EGFR PPI strengths measured for EVs generally show a tight correlation with those determined for the original cells. The results suggest that EGFRs contained in EVs closely reflect the cellular EGFR in terms of their downstream signaling capacity.

Structural and biochemical insights into the role of testis-expressed gene 14 (TEX14) in forming the stable intercellular bridges of germ cells.

Intercellular bridges are a conserved feature of spermatogenesis in mammalian germ cells and derive from arresting cell abscission at the final stage of cytokinesis. However, it remains to be fully understood how germ cell abscission is arrested in the presence of general cytokinesis components. The TEX14 (testis-expressed gene 14) protein is recruited to the midbody and plays a key role in the inactivation of germ cell abscission. To gain insights into the structural organization of TEX14 at the midbody, we have determined the crystal structures of the EABR [endosomal sorting complex required for transport (ESCRT) and ALIX-binding region] of CEP55 bound to the TEX14 peptide (or its chimeric peptides) and performed functional characterization of the CEP55-TEX14 interaction by multiexperiment analyses. We show that TEX14 interacts with CEP55-EABR via its AxGPPx3Y (Ala793, Gly795, Pro796, Pro797, and Tyr801) and PP (Pro803 and Pro804) sequences, which together form the AxGPPx3YxPP motif. TEX14 competitively binds to CEP55-EABR to prevent the recruitment of ALIX, which is a component of the ESCRT machinery with the AxGPPx3Y motif. We also demonstrate that a high affinity and a low dissociation rate of TEX14 to CEP55, and an increase in the local concentration of TEX14, cooperatively prevent ALIX from recruiting ESCRT complexes to the midbody. The action mechanism of TEX14 suggests a scheme of how to inactivate the abscission of abnormal cells, including cancer cells.

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 µM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2α.

Nucleolar phosphoprotein 140 (Nopp140) is a nucleolar protein, more than 80% of which is disordered. Previous studies have shown that the C-terminal region of Nopp140 (residues 568-596) interacts with protein kinase CK2α, and inhibits the catalytic activity of CK2. Although the region of Nopp140 responsible for the interaction with CK2α was identified, the structural features and the effect of this interaction on the structure of Nopp140 have not been defined due to the difficulty of structural characterization of disordered protein. In this study, the disordered feature of Nopp140 and the effect of CK2α on the structure of Nopp140 were examined using single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR). The interaction with CK2α was increased conformational rigidity of the CK2α-interacting region of Nopp140 (Nopp140C), suggesting that the disordered and flexible conformation of Nopp140C became more rigid conformation as it binds to CK2α. In addition, site specific spin labeling and EPR analysis confirmed that the residues 574-589 of Nopp140 are critical for binding to CK2α. Similar technical approaches can be applied to analyze the conformational changes in other IDPs during their interactions with binding partners.

Dynamic light scattering analysis of SNARE-driven membrane fusion and the effects of SNARE-binding flavonoids.

Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.

Beta-amyloid oligomers activate apoptotic BAK pore for cytochrome c release.

In Alzheimer's disease, cytochrome c-dependent apoptosis is a crucial pathway in neuronal cell death. Although beta-amyloid (Aß) oligomers are known to be the neurotoxins responsible for neuronal cell death, the underlying mechanisms remain largely elusive. Here, we report that the oligomeric form of synthetic Aß of 42 amino acids elicits death of HT-22 cells. But, when expression of a bcl-2 family protein BAK is suppressed by siRNA, Aß oligomer-induced cell death was reduced. Furthermore, significant reduction of cytochrome c release was observed with mitochondria isolated from BAK siRNA-treated HT-22 cells. Our in vitro experiments demonstrate that Aß oligomers bind to BAK on the membrane and induce apoptotic BAK pores and cytochrome c release. Thus, the results suggest that Aß oligomers function as apoptotic ligands and hijack the intrinsic apoptotic pathway to cause unintended neuronal cell death.

Extracellular Vesicles in Therapeutics: A Comprehensive Review on Applications, Challenges, and Clinical Progress.

Small Extracellular Vesicles (sEVs) are typically 30-150 nm in diameter, produced inside cells, and released into the extracellular space. These vesicles carry RNA, DNA, proteins, and lipids that reflect the characteristics of their parent cells, enabling communication between cells and the alteration of functions or differentiation of target cells. Owing to these properties, sEVs have recently gained attention as potential carriers for functional molecules and drug delivery tools. However, their use as a therapeutic platform faces limitations, such as challenges in mass production, purity issues, and the absence of established protocols and characterization methods. To overcome these, researchers are exploring the characterization and engineering of sEVs for various applications. This review discusses the origins of sEVs and their engineering for therapeutic effects, proposing areas needing intensive study. It covers the use of cell-derived sEVs in their natural state and in engineered forms for specific purposes. Additionally, the review details the sources of sEVs and their subsequent purification methods. It also outlines the potential of therapeutic sEVs and the requirements for successful clinical trials, including methods for large-scale production and purification. Finally, we discuss the progress of ongoing clinical trials and the implications for future healthcare, offering a comprehensive overview of the latest research in sEV applications.

Novel Personalized Cancer Vaccine Using Tumor Extracellular Vesicles with Attenuated Tumorigenicity and Enhanced Immunogenicity.

Cancer vaccines offer a promising avenue in cancer immunotherapy by inducing systemic, tumor-specific immune responses. Tumor extracellular vesicles (TEVs) are nanoparticles naturally laden with tumor antigens, making them appealing for vaccine development. However, their inherent malignant properties from the original tumor cells limit their direct therapeutic use. This study introduces a novel approach to repurpose TEVs as potent personalized cancer vaccines. The study shows that inhibition of both YAP and autophagy not only diminishes the malignancy-associated traits of TEVs but also enhances their immunogenic attributes by enriching their load of tumor antigens and adjuvants. These revamped TEVs, termed attenuated yet immunogenically potentiated TEVs (AI-TEVs), showcase potential in inhibiting tumor growth, both as a preventive measure and a possible treatment for recurrent cancers. They prompt a tumor-specific and enduring immune memory. In addition, by showing that AI-TEVs can counteract cancer growth in a personalized vaccine approach, a potential strategy is presented for developing postoperative cancer immunotherapy that's enduring and tailored to individual patients.

Novel insights into paclitaxel's role on tumor-associated macrophages in enhancing PD-1 blockade in breast cancer treatment.

BACKGROUND: Triple-negative breast cancer (TNBC) poses unique challenges due to its complex nature and the need for more effective treatments. Recent studies showed encouraging outcomes from combining paclitaxel (PTX) with programmed cell death protein-1 (PD-1) blockade in treating TNBC, although the exact mechanisms behind the improved results are unclear. METHODS: We employed an integrated approach, analyzing spatial transcriptomics and single-cell RNA sequencing data from TNBC patients to understand why the combination of PTX and PD-1 blockade showed better response in TNBC patients. We focused on toll-like receptor 4 (TLR4), a receptor of PTX, and its role in modulating the cross-presentation signaling pathways in tumor-associated macrophages (TAMs) within the tumor microenvironment. Leveraging insights obtained from patient-derived data, we conducted in vitro experiments using immunosuppressive bone marrow-derived macrophages (iBMDMs) to validate if PTX could augment the cross-presentation and phagocytosis activities. Subsequently, we extended our study to an in vivo murine model of TNBC to ascertain the effects of PTX on the cross-presentation capabilities of TAMs and its downstream impact on CD8+ T cell-mediated immune responses. RESULTS: Data analysis from TNBC patients revealed that the activation of TLR4 and cross-presentation signaling pathways are crucial for the antitumor efficacy of PTX. In vitro studies showed that PTX treatment enhances the cross-presentation ability of iBMDMs. In vivo experiments demonstrated that PTX activates TLR4-dependent cross-presentation in TAMs, improving CD8+ T cell-mediated antitumor responses. The efficacy of PTX in promoting antitumor immunity was elicited when combined with PD-1 blockade, suggesting a complementary interaction. CONCLUSIONS: This study reveals how PTX boosts the effectiveness of PD-1 inhibitors in treating TNBC. We found that PTX activates TLR4 signaling in TAMs. This activation enhances their ability to present antigens, thereby boosting CD8+ T cell antitumor responses. These findings not only shed light on PTX's immunomodulatory role in TNBC but also underscore the potential of targeting TAMs' antigen presentation capabilities in immunotherapy approaches.

Identification of the inhibitory mechanism of ecumicin and rufomycin 4-7 on the proteolytic activity of Mycobacterium tuberculosis ClpC1/ClpP1/ClpP2 complex.

Ecumicin and rufomycin 4-7 disrupt protein homeostasis in Mycobacterium tuberculosis by inhibiting the proteolytic activity of the ClpC1/ClpP1/ClpP2 complex. Although these compounds target ClpC1, their effects on the ATPase activity of ClpC1 and proteolytic activity of ClpC1/ClpP1/ClpP2 vary. Herein, we explored the ClpC1 molecular dynamics with these compounds through fluorescence correlation spectroscopy. The effect of these compounds on the ATPase activity of ClpC1-cys, the recombinant protein for fluorescence labeling, and proteolytic activity of ClpC1-cys/ClpP1/ClpP2 were identical to those of native ClpC1, whereas the intermolecular dynamics of fluorescence-labelled ClpC1 were different. Treatment with up to 1 nM ecumicin increased the population of slower diffused ClpC1 components compared with ClpC1 without ecumicin. However, this population was considerably reduced when treated with 10 nM ecumicin. Rufomycin 4-7 treatment resulted in a slower diffused component of ClpC1, and the portion of this component increased in a concentration-dependent manner. Ecumicin can generate an abnormal ClpC1 component, which cannot form normal ClpC1/ClpP1/ClpP2, via two different modes. Rufomycin 4-7 only generates slower diffused ClpC1 component that is inadequate to form normal ClpC1/ClpP1/ClpP2. Overall, we demonstrate that ecumicin and rufomycin 4-7 use different action mechanisms to generate abnormal ClpC1 components that cannot couple with ClpP1/ClpP2.

Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability.

Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).

Effects of Transcription-Dependent Physical Perturbations on the Chromosome Dynamics in Living Cells.

Recent studies with single-particle tracking in live cells have revealed that chromatin dynamics are directly affected by transcription. However, how transcription alters the chromatin movements followed by changes in the physical properties of chromatin has not been elucidated. Here, we measured diffusion characteristics of chromatin by targeting telomeric DNA repeats with CRISPR-labeling. We found that transcription inhibitors that directly block transcription factors globally increased the movements of chromatin, while the other inhibitor that blocks transcription by DNA intercalating showed an opposite effect. We hypothesized that the increased mobility of chromatin by transcription inhibition and the decreased chromatin movement by a DNA intercalating inhibitor is due to alterations in chromatin rigidity. We also tested how volume confinement of nuclear space affects chromatin movements. We observed decreased chromatin movements under osmotic pressure and with overexpressed chromatin architectural proteins that compact chromatin.

Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude.

How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

Advantage of extracellular vesicles in hindering the CD47 signal for cancer immunotherapy.

The cluster of differentiation 47 (CD47) protein is abundantly expressed on various malignant cells and suppresses the phagocytic function of macrophages and dendritic cells. High CD47 expression levels are correlated with poor cancer survival. Antagonizing CD47 antibodies with potent antitumor effects have been developed in clinical trials, but have critical side effects, inducing anemia and thrombocytopenia. To develop a safe and potent CD47 blockade, we designed extracellular vesicles (EVs) harboring signal regulatory protein alpha (SIPRα)-EV-SIRPα (EVs that express SIPRα). EV-SIRPα showed minimal toxic effects on hematologic parameters and utilized RBCs as delivery vehicles to tumors rather than inducing anemia. EV-SIRPα inhibited ligation of residual CD47 molecules, which attribute to the EV-endocytosis-mediated CD47 depletion and steric hindrance of EV. In an immunologically cold tumor model, EV-SIRPα induced tumor-specific T-cell-mediated antitumor effects. When directly administered to the accessible lesions, EV-SIRPα monotherapy elicited an abscopal effect in the B16F10 tumor model by increasing immune cell infiltration and CD8+-mediated immunity against non-treated tumors. The combinational approach by loading doxorubicin into the EV-SIRPα dramatically reduced the tumor burden and led to 80% complete remission rate. Thus, a potent EV-based CD47 blockade that is hematologically safe, has efficient signaling blocking efficacy, and has systemic antitumor immunity against cancer is recommended.

Water meniscus-directed organization of liquid-ordered domains in lipid monolayer.

We report on a spatial organization process of liquid-ordered (l(o)) domains in a lipid monolayer into a two dimensional array on structurally patterned substrates. The curvature of a lipid monolayer which strongly obeys the water meniscus at an air-water interface is manipulated to create two different, curved and flat, zones during the water evaporation in the structural patterns. Due to the high bending rigidity, l(o) domains are diffused into the flat region to avoid the elastic deformation. The Helfrich-type thermodynamic criterion for coarsening of l(o) domains was first presented and confirmed through experimental results obtained in various patterns. Our topographic system will be very useful for understanding a fundamental concept as well as a quantitative criterion for curvature-driven organization of l(o) domains in biological membranes.

Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field.

The quantum yield of a fluorophore is reduced when two or more identical fluorophores are in close proximity to each other. The study of protein folding or particle aggregation is can be done based on this above-mentioned phenomenon-called self-quenching. However, it is challenging to characterize the self-quenching of a fluorophore at high concentrations because of the inner filter effect, which involves depletion of excitation light and re-absorption of emission light. Herein, a novel method to directly evaluate the self-quenching behavior of fluorophores was developed. The evanescent field from an objective-type total internal reflection fluorescence (TIRF) microscope was used to reduce the path length of the excitation and emission light to ~100 nm, thereby supressing the inner filter effect. Fluorescence intensities of sulforhodamine B, fluorescein isothiocyanate (FITC), and calcein solutions with concentrations ranging from 1 µM to 50 mM were directly measured to evaluate the concentration required for 1000-fold degree of self-quenching and to examine the different mechanisms through which the fluorophores undergo self-quenching.

In situ immunogenic clearance induced by a combination of photodynamic therapy and rho-kinase inhibition sensitizes immune checkpoint blockade response to elicit systemic antitumor immunity against intraocular melanoma and its metastasis.

BACKGROUND: Uveal melanoma (UM) is the most frequent intraocular malignancy and is resistant to immunotherapy. Nearly 50% of patients with UM develop metastatic disease, and the overall survival outcome remains very poor. Therefore, a treatment regimen that simultaneously targets primary UM and prevents metastasis is needed. Here, we suggest an immunotherapeutic strategy for UM involving a combination of local photodynamic therapy (PDT), rho-kinase (ROCK) inhibitor, and PD-1/PD-L1 immune checkpoint blockade. METHODS: The antitumor efficacy and immune response of monotreatment or combinational treatment were evaluated in B16F10-bearing syngeneic mouse models. Abscopal antitumor immune responses induced by triple-combinational treatment were validated in syngeneic bilateral B16F10 models. After each treatment, the immune profiles and functional examinations were assessed in tumors and tumor draining lymph nodes by flow cytometry, ELISA, and immunofluorescence assays. In orthotopic intraocular melanoma models, the location of the immune infiltrate in the tumor microenvironment (TME) was evaluated after each treatment by multiplex immunohistochemistry and metastatic nodules were monitored. RESULTS: PDT with Ce6-embedded nanophotosensitizer (FIC-PDT) elicited immunogenic cell death and stimulated antigen-presenting cells. In situ immunogenic clearance induced by a combination of FIC-PDT with ripasudil, a clinically approved ROCK inhibitor, stimulated antigen-presenting cells, which in turn primed tumor-specific cytotoxic T cells. Moreover, local immunogenic clearance sensitized PD-1/PD-L1 immune checkpoint blockade responses to reconstruct the TME immune phenotypes of cold tumors into hot tumors, resulting in recruitment of robust cytotoxic CD8+ T cells in the TME, propagation of systemic antitumor immunity to mediate abscopal effects, and prolonged survival. In an immune-privileged orthotopic intraocular melanoma model, even low-dose FIC-PDT and ripasudil combined with anti-PD-L1 antibody reduced the primary tumor burden and prevented metastasis. CONCLUSIONS: A combination of localized FIC-PDT and a ROCK inhibitor exerted a cancer vaccine-like function. Immunogenic clearance led to the trafficking of CD8+ T cells into the primary tumor site and sensitized the immune checkpoint blockade response to evoke systemic antitumor immunity to inhibit metastasis, one of the major challenges in UM therapy. Thus, immunogenic clearance induced by FIC-PDT and ROCK inhibitor combined with anti-PD-L1 antibody could be a potent immunotherapeutic strategy for UM.