MHC class I-dressing is mediated via phosphatidylserine recognition and is enhanced by polyI:C.

In addition to cross-presentation, cross-dressing plays an important role in the induction of CD8+ T cell immunity. In the process of cross-dressing, conventional dendritic cells (DCs) acquire major histocompatibility complex class I (MHCI) from other cells and subsequently prime CD8+ T cells via the pre-formed antigen-MHCI complexes without antigen processing. However, the mechanisms underlying the cross-dressing pathway, as well as the relative contributions of cross-presentation and cross-dressing to CD8+ T cell priming are not fully understood. Here, we demonstrate that DCs rapidly acquire MHCI-containing membrane fragments from dead cells via the phosphatidylserine recognition-dependent mechanism for cross-dressing. The MHCI dressing is enhanced by a TLR3 ligand polyinosinic-polycytidylic acid (polyI:C). Further, polyI:C promotes not only cross-presentation but also cross-dressing in vivo. Taken together, these results suggest that cross-dressing as well as cross-presentation is involved in inflammatory diseases associated with cell death and type I IFN production.

Extracellular vesicles promote silica nanoparticle aggregation that inhibits silica-induced cytotoxicity.

Amorphous silica has been approved as a food and pharmaceutical additive. However, its potential to enhance the carcinogenicity of epithelial cells is incontrovertible. With their expanded surface area per unit mass and distinctive cellular incorporation, nano-sized silica particles (nSPs) exhibit heightened cytotoxicity compared to micrometer-sized counterparts. The precise effect of nSPs on the generation of small extracellular vesicles (sEVs) within endosomes after cellular uptake remains unclear. In the present study, we explored the secretion of sEVs from cells and their functional implications following exposure to nSPs. Our findings demonstrate that nSP50 exposure not only induced epithelial-mesenchymal transition (EMT) but also promoted the maturation of multivesicular endosomes (MVEs) along with the secretion of sEVs in A549 cells. Inhibition of sEV secretion using GW4869 and apoptosis activator 2 exacerbated nSP50-induced EMT, indicating that sEV secretion may suppress EMT. Analysis of the function of sEV in a cell-free system revealed that co-incubation of sEVs with nSP50 led to the formation of micrometer-sized aggregates, which exhibited limited uptake efficiency within A549 cells. These results strongly suggest that the secretion of sEVs plays a protective role against the cytotoxicity attributed to nSP50 exposure.

Precise analysis of single small extracellular vesicles using flow cytometry.

Methods that enable specific and sensitive quantification of small extracellular vesicles (sEVs) using flow cytometry are still under development. Aggregation or adsorption of antibodies causes sub-nano sized particles or non-specific binding and largely affects the results of flow cytometric analysis of single sEVs. Comparison of control IgG and target-specific IgG is inappropriate because they have different characters. Here, we evaluate four preparation methods for flow cytometry, including ultracentrifugation, density gradient centrifugation, size exclusion chromatography (SEC), and the TIM4-affinity method by using tetraspanin-deficient sEVs. The ultracentrifugation or density gradient centrifugation preparation method has large false-positive rates for tetraspanin staining. Conversely, preparation methods using SEC or the TIM4-affinity method show specific detection of single sEVs, which elucidate the roles of sEV biogenesis regulators in the generation of sEV subpopulations. The methods are also useful for the detection of rare disease-related markers, such as PD-L1. Flow cytometric analysis using SEC or the TIM4-affinity method could accelerate research into sEV biogenesis and the development of sEV-based diagnostics and therapies.

Peritumoral SPARC expression induced by exosomes from nasopharyngeal carcinoma infected Epstein-Barr virus: A poor prognostic marker.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) cells have high metastatic potential. Recent research has revealed that the interaction of between tumor cells and the surrounding stroma plays an important role in tumor invasion and metastasis. In this study, we showed the prognostic value of expression of SPARC, an extracellular matrix protein with multiple cellular functions, in normal adjacent tissues (NAT) surrounding NPC. In the immunohistochemical analysis of 51 NPC biopsy specimens, SPARC expression levels were significantly elevated in the NAT of EBER (EBV-encoded small RNA)-positive NPC compared to that in the NAT of EBER-negative NPC. Moreover, increased SPARC expression in NAT was associated with a worsening of overall survival. The enrichment analysis of RNA-seq of publicly available NPC and NAT surrounding NPC data showed that high SPARC expression in NPC was associated with epithelial mesenchymal transition promotion, and there was a dynamic change in the gene expression profile associated with interference of cellular proliferation in NAT, including SPARC expression. Furthermore, EBV-positive NPC cells induce SPARC expression in normal nasopharyngeal cells via exosomes. Induction of SPARC in cancer-surrounding NAT cells reduced intercellular adhesion in normal nasopharyngeal structures and promoted cell competition between cancer cells and normal epithelial cells. These results suggest that epithelial cells loosen their own binding with the extracellular matrix as well as stromal cells, facilitating the invasion of tumor cells into the adjacent stroma by activating cell competition. Our findings reveal a new mechanism by which EBV creates a pro-metastatic microenvironment by upregulating SPARC expression in NPC.

Nanopipette Fabrication Guidelines for SICM Nanoscale Imaging.

Scanning ion conductance microscopy (SICM) is a promising tool for visualizing the dynamics of nanoscale cell surface topography. However, there are still no guidelines for fabricating nanopipettes with ideal shape consisting of small apertures and thin glass walls. Therefore, most of the SICM imaging has been at a standstill at the submicron scale. In this study, we established a simple and highly reproducible method for the fabrication of nanopipettes with sub-20 nm apertures. To validate the improvement in the spatial resolution, we performed time-lapse imaging of the formation and disappearance of endocytic pits as a model of nanoscale time-lapse topographic imaging. We have also successfully imaged the localization of the hot spot and the released extracellular vesicles. The nanopipette fabrication guidelines for the SICM nanoscale topographic imaging can be an essential tool for understanding cell-cell communication.

A validation study for the utility of serum microRNA as a diagnostic and prognostic marker in patients with osteosarcoma.

In our previous study, osteosarcoma advanced locally, and metastasis was promoted through the secretion of large number of small extracellular vesicles, followed by suppressing osteoclastogenesis via the upregulation of microRNA (miR)-146a-5p. An additional 12 miRNAs in small extracellular vesicles were also detected ≥6× as frequently in high-grade malignancy with the capacity to metastasize as in those with a low metastatic potential. However, the utility of these 13 miRNAs for determining the prognosis or diagnosis of osteosarcoma has not been validated in the clinical setting. In the present study, the utility of these miRNAs as prognostic and diagnostic markers was therefore assessed. In total, 30 patients with osteosarcoma were retrospectively reviewed, and the survival rate was compared according to the serum miRNA levels in 27 patients treated with chemotherapy and surgery. In addition, to confirm diagnostic competency for osteosarcoma, the serum miRNA levels were compared with those in patients with other bone tumors (n=112) and healthy controls (n=275). The patients with osteosarcoma with high serum levels of several miRNAs (miR-146a-5p, miR-1260a, miR-487b-3p, miR-1260b and miR-4758-3p) exhibited an improved survival rate compared with those with low levels. In particular, patients with high serum levels of miR-1260a exhibited a significantly improved overall survival rate, metastasis-free survival rate and disease-free survival rate compared with those with low levels. Thus, serum miR-1260a may potentially be a prognostic marker for patients with osteosarcoma. Moreover, patients with osteosarcoma had higher serum miR-1261 levels than those with benign or intermediate-grade bone tumors and thus may be a potential therapeutic target, in addition to being useful for differentiating whether or not a bone tumor is high-grade. A larger investigation is required to clarify the actual utility of these miRNAs in the clinical setting.

Nanoscopic Assessment of Anti-SARS-CoV-2 Spike Neutralizing Antibody Using High-Speed AFM.

Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.

Preventing SARS-CoV-2 Infection Using Anti-spike Nanobody-IFN-ß Conjugated Exosomes.

PURPOSE: To inhibit the transmission of SARS-CoV-2, we developed engineered exosomes that were conjugated with anti-spike nanobodies and type I interferon ß (IFN-ß). We evaluated the efficacy and potency of nanobody-IFN-ß conjugated exosomes to treatment of SARS-CoV-2 infection. METHODS: Milk fat globule epidermal growth factor 8 (MFG-E8) is a glycoprotein that binds to phosphatidylserine (PS) exposed on the exosomes. We generated nanobody-IFN-ß conjugated exosomes by fusing an anti-spike nanobody and IFN-ß with MFG-E8. We used the SARS-CoV-2 pseudovirus with the spike of the D614G mutant that encodes ZsGreen to mimic the infection process of the SARS-CoV-2. The SARS-CoV-2 pseudovirus was infected with angiotensin-converting enzyme-2 (ACE2) expressing adenocarcinomic human alveolar basal epithelial cells (A549) or ACE2 expressing HEK-blue IFNα/ß cells in the presence of nanobody-IFN-ß conjugated exosomes. By assessing the expression of ZsGreen in target cells and the upregulation of interferon-stimulated genes (ISGs) in infected cells, we evaluated the anti-viral effects of nanobody-IFN-ß conjugated exosomes. RESULTS: We confirmed the anti-spike nanobody and IFN-ß expressions on the exosomes. Exosomes conjugated with nanobody-hIFN-ß inhibited the interaction between the spike protein and ACE2, thereby inhibiting the infection of host cells with SARS-CoV-2 pseudovirus. At the same time, IFN-ß was selectively delivered to SARS-CoV-2 infected cells, resulting in the upregulation of ISGs expression. CONCLUSION: Exosomes conjugated with nanobody-IFN-ß may provide potential benefits in the treatment of COVID-19 because of the cooperative anti-viral effects of the anti-spike nanobody and the IFN-ß.

Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical stresses revealed by HS-AFM.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

TIM4-Affinity Methods Targeting Phosphatidylserine for Isolation or Detection of Extracellular Vesicles.

Small extracellular vesicles (SEVs) secreted from various cells are lipid bilayer vesicles, 30-150 nm in size, that carry proteins, nucleic acids, and lipids as cargos to other cells. They include exosomes, which are generated in multivesicular endosomes (MVEs) and secreted upon fusion of MVEs with plasma membranes and a part of microvesicles, which directly bud from plasma membranes. SEVs have attracted attention as diagnostic and drug discovery targets, since it has been demonstrated that SEVs are involved in the intercellular communication in many diseases and physiological phenomena such as cancer, neurodegenerative diseases, and immunity. There are five isolation methods for SEVs, which include ultracentrifugation, density gradient ultracentrifugation, polymer precipitation, affinity isolation, and size-exclusion chromatography. The affinity isolation, which isolates SEVs using magnetic beads conjugated with binding molecules such as antibodies, has the ability to isolate highly pure SEVs in character. However, the population of SEVs is limited by the binding molecules and it is difficult to elute intact SEVs from the antibody beads. In this chapter, we present a TIM4-affinity isolation method that targets phosphatidylserine (PS), a component of the SEV membrane. TIM4 binds to PS in a Ca2+-dependent manner, which enables the elution of intact SEVs from TIM4-beads in the presence of the chelating reagent ethylenediaminetetraacetic acid (EDTA). The TIM4-affinity isolation method helps overcome the disadvantages of the affinity isolation method and enables the isolation of heterogeneous SEVs at high purity. This method will facilitate the functional analysis of SEVs, development of diagnostic methods, and drug development of engineered SEVs.

Extracellular Vesicles Contribute to the Metabolism of Transthyretin Amyloid in Hereditary Transthyretin Amyloidosis.

Hereditary (variant) transthyretin amyloidosis (ATTRv amyloidosis), which is caused by variants in the transthyretin (TTR) gene, leads to TTR amyloid deposits in multiple organs and various symptoms such as limb ataxia, muscle weakness, and cardiac failure. Interaction between amyloid proteins and extracellular vesicles (EVs), which are secreted by various cells, is known to promote the clearance of the proteins, but it is unclear whether EVs are involved in the formation and deposition of TTR amyloid in ATTRv amyloidosis. To clarify the relationship between ATTRv amyloidosis and EVs, serum-derived EVs were analyzed. In this study, we showed that cell-derived EVs are involved in the formation of TTR amyloid deposits on the membrane of small EVs, as well as the deposition of TTR amyloid in cells. Human serum-derived small EVs also altered the degree of aggregation and deposition of TTR. Furthermore, the amount of TTR aggregates in serum-derived small EVs in patients with ATTRv amyloidosis was lower than that in healthy controls. These results indicate that EVs contribute to the metabolism of TTR amyloid, and suggest that TTR in serum-derived small EVs is a potential target for future ATTRv amyloidosis diagnosis and therapy.

Millisecond dynamic of SARS-CoV-2 spike and its interaction with ACE2 receptor and small extracellular vesicles.

SARS-CoV-2 spike protein (S) binds to human angiotensin-converting enzyme 2 (hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we use high-speed atomic force microscopy (HS-AFM) for real-time visualization of S, and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show conformational heterogeneity of S, flexibility of S stalk and receptor-binding domain (RBD), and pH/temperature-induced conformational change of S. S in an S-ACE2 complex appears as an all-RBD up conformation. The complex acquires a distinct topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S-hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2-expressing sEVs for viral neutralization. In contrary, S2 subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking the viral entry scenario. Altogether, our study provides a platform that is suitable for real-time visualization of various entry inhibitors, neutralizing antibodies, and sEV-based decoy in blocking viral entry. Teaser: Comprehensive observation of SARS-CoV-2 spike and its interaction with receptor ACE2 and sEV-based decoy in real time using HS-AFM.

Identification of small compounds regulating the secretion of extracellular vesicles via a TIM4-affinity ELISA.

Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell-cell communication by transporting proteins, lipids, and nucleic acids. As the involvement of EVs in diseases has become apparent, druggable regulators of EV secretion are required. However, the lack of a highly sensitive EV detection system has made the development of EV regulators difficult. We developed an ELISA system using a high-affinity phosphatidylserine-binder TIM4 to capture EVs and screened a 1567-compound library. Consequently, we identified one inhibitor and three activators of EV secretion in a variety of cells. The inhibitor, apoptosis activator 2, suppressed EV secretion via a different mechanism and had a broader cellular specificity than GW4869. Moreover, the three activators, namely cucurbitacin B, gossypol, and obatoclax, had broad cellular specificity, including HEK293T cells and human mesenchymal stem cells (hMSCs). In vitro bioactivity assays revealed that some regulators control EV secretion from glioblastoma and hMSCs, which induces angiogenesis and protects cardiomyocytes against apoptosis, respectively. In conclusion, we developed a high-throughput method to detect EVs with high sensitivity and versatility, and identified four compounds that can regulate the bioactivity of EVs.

Osteosarcoma-Derived Small Extracellular Vesicles Enhance Tumor Metastasis and Suppress Osteoclastogenesis by miR-146a-5p.

Osteosarcoma is the most frequent type of primary bone tumor in children and adolescents, thus care for patients with malignant osteosarcoma is strongly required. The roles of small extracellular vesicles (SEVs) in enhancing metastases have been demonstrated in multiple tumors, but they are still poorly understood in osteosarcoma. Hence, this study investigated the effects of SEVs on progression and the tumor microenvironment in mice and patients. In an orthotopic implantation study, we found that osteosarcoma-derived SEVs had the potential to enhance metastases and angiogenesis. In addition, osteosarcoma-derived SEVs decreased the number of mature osteoclasts in vivo. In vitro osteoclastogenesis studies revealed that the inhibition of osteoclast maturation by osteosarcoma-derived SEVs was mediated by suppressing the NF-κB signal pathway. MicroRNA analysis of SEVs from different malignant human osteosarcomas revealed that miR-146a-5p was involved in the inhibition of osteoclastogenesis. In osteosarcoma patients, lower numbers of osteoclasts in biopsy specimens at the first visits were correlated with higher malignancy. These findings indicated that osteosarcoma-derived SEVs enhance distant metastasis of osteosarcomas by inhibiting osteoclast maturation, which may be a useful prognostic marker. This diagnostic method may enable to predict malignancy at early stage, and help to provide optimal care to patients with risk of high malignancy.

Structural and mechanical characteristics of exosomes from osteosarcoma cells explored by 3D-atomic force microscopy.

Exosomes have recently gained interest as mediators of cell-to-cell communication and as potential biomarkers for cancer and other diseases. They also have potential as nanocarriers for drug delivery systems. Therefore, detailed structural, molecular, and biomechanical characterization of exosomes is of great importance for developing methods to detect and identify the changes associated with the presence of cancer and other diseases. Here, we employed three-dimensional atomic force microscopy (3D-AFM) to reveal the structural and nanomechanical properties of exosomes at high spatial resolution in physiologically relevant conditions. The substructural details of exosomes released from three different cell types were determined based on 3D-AFM force mapping. The resulting analysis revealed the presence of distinct local domains bulging out from the exosome surfaces, which were associated with the exosomal membrane proteins present on the outer surface. The nanomechanical properties of individual exosomes were determined from the 3D-force maps. We found a considerably high elastic modulus, ranging from 50 to 350 MPa, as compared to that obtained for synthetic liposomes. Moreover, malignancy-dependent changes in the exosome mechanical properties were revealed by comparing metastatic and nonmetastatic tumor cell-derived exosomes. We found a clear difference in their Young's modulus values, suggesting differences in their protein profiles and other exosomal contents. Exosomes derived from a highly aggressive and metastatic k-ras-activated human osteosarcoma (OS) cell line (143B) showed a higher Young's modulus than that derived from a nonaggressive and nonmetastatic k-ras-wildtype human OS cell line (HOS). The increased elastic modulus of the 143B cell-derived exosomes was ascribed to the presence of abundant specific proteins responsible for elastic fiber formation as determined by mass spectroscopy and confirmed by western blotting and ELISA. Therefore, we conclude that exosomes derived from metastatic tumor cells carry an exclusive protein content that differs from their nonmetastatic counterparts, and thus they exhibit different mechanical characteristics. Discrimination between metastatic and nonmetastatic malignant cell-derived exosomes would be of great importance for studying exosome biological functions and using them as diagnostic biomarkers for various tumor types. Our findings further suggest that metastatic tumor cells release exosomes that express increased levels of elastic fiber-associated proteins to preserve their softness.

Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway.

Accumulating evidence indicates the presence of cytoplasmic DNAs in various types of malignant cells, and its involvement in anti-cancer drug- or radiotherapy-mediated DNA damage response and replication stress. However, the pathophysiological roles of cytoplasmic DNAs in leukemias remain largely unknown. We observed that during hematopoietic stem cell transplantation (HSCT) in mouse myeloid leukemia models, double-stranded (ds)DNAs were constitutively secreted in the form of extracellular vesicles (EVs) from myeloid leukemia cells and were transferred to the donor cells to dampen their hematopoietic capabilities. Subsequent analysis of cytoplasmic DNA dynamics in leukemia cells revealed that autophagy regulated cytoplasmic dsDNA accumulation and subsequent redistribution into EVs. Moreover, accumulated cytoplasmic dsDNAs activated STING pathway, thereby reducing leukemia cell viability through reactive oxygen species (ROS) generation. Pharmaceutical inhibition of autophagosome formation induced cytoplasmic DNA accumulation, eventually triggering cytoplasmic DNA sensing pathways to exert cytotoxicity, preferentially in leukemia cells. Thus, manipulation of cytoplasmic dsDNA dynamics can be a novel and potent therapeutic strategy for myeloid leukemias.

Characterization of LILRB3 and LILRA6 allelic variants in the Japanese population.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) are encoded by members of a human multigene family, comprising 11 protein-coding genes and two pseudogenes. Among the LILRs, LILRB3 and LILRA6 show the highest homology with each other, along with high allelic and copy number variations. Therefore, it has been difficult to discriminate between them, both genetically and functionally, precluding disease association studies of LILRB3 and LILRA6. In this study, we carefully performed variant screening of LILRB3 and LILRA6 by cDNA cloning from Japanese individuals and identified four allelic lineages showing significantly high non-synonymous-to-synonymous ratios in pairwise comparisons. Furthermore, the extracellular domains of the LILRB3*JP6 and LILRA6*JP1 alleles were identical at the DNA level, suggesting that gene conversion-like events diversified LILRB3 and LILRA6. To determine the four allelic lineages from genomic DNA, we established a lineage typing method that accurately estimated the four allelic lineages in addition to specific common alleles from genomic DNA. Analysis of LILRA6 copy number variation revealed one, two, and three copies of LILRA6 in the Japanese-in-Tokyo (JPT) population. Flow cytometric analysis showed that an anti-LILRB3 antibody did not recognize the second most common lineage in the Japanese population, indicating significant amino acid differences across the allelic lineages. Taken together, our findings indicate that our lineage typing is useful for classifying the lineage-specific functions of LILRB3 and LILRA6, serving as the basis for disease association studies.

Tumor-secreted proliferin-1 regulates adipogenesis and lipolysis in cachexia.

Cancer-associated cachexia (CAC) is a common syndrome in cancer patients and is characterized by loss of body weight accompanied by the atrophy of fat and skeletal muscle. Metabolic changes are a critical factor in CAC; however, the mechanisms through which tumors inhibit adipogenesis and promote lipolysis are poorly understood. To clarify these mechanisms, we investigated adipogenesis-limiting factors released by tumors in a cell culture system. We identified proliferin-1 (PLF-1), a member of the growth hormone/prolactin gene family, as a key factor secreted from certain tumors that inhibited preadipocyte maturation and promoted the lipolysis of mature adipocytes. Importantly, mice transplanted with PLF-1-depleted tumor cells were protected from fat loss due to CAC. These data show that tumor-secreted PLF-1 plays an essential role in impaired adipogenesis and accelerated lipolysis and is a potential therapeutic target against CAC.

Emerging roles of extracellular vesicles in physiology and disease.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are small membrane vesicles secreted by almost all cell types and are abundant in blood, body fluids, such as urine, spinal fluid, tears and saliva, and cell culture media. From an evolutionary perspective, they are biologically significant as a means for expelling unwanted cellular contents. Recently, EVs have received considerable attention as messengers of intercellular communication networks, allowing the exchange of proteins and lipids between the cells producing them and target cells that trigger various cellular responses. EVs also carry mRNAs and microRNAs inside them, transferring genetic information among cells. In addition, the expression pattern of these molecules is related to the cellular state and the progression of diseases, and the search for biomarkers within the EV is underway in many research fields. However, the physiological and pathophysiological roles of EVs remain largely elusive. Therefore, in this special issue, we have compiled reviews of the latest research findings on EV research.

Transient IGF-1R inhibition combined with osimertinib eradicates AXL-low expressing EGFR mutated lung cancer.

Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC.