Noriko Gotoh

The functional accessibility of membrane proteins is a fundamental determinant of biological activity but cannot be accurately inferred from protein abundance alone. Current extracellular vesicle (EV) analyses primarily quantify expression levels, providing limited information on whether surface ligands are physically accessible for molecular interactions. Here, we establish high‐speed atomic force microscopy (HS‐AFM) as a label‐free, real‐time approach for directly visualizing and quantifying ligand accessibility on individual small extracellular vesicles (sEVs) under near‐physiological conditions. Using programmed death‐ligand 1 (PD‐L1) as a model immune checkpoint, we characterize antibody binding dynamics on sEVs derived from luminal breast cancer cells, triple‐negative breast cancer (TNBC) cells, and patient‐derived TNBC cells. PD‐L1‐high TNBC sEVs exhibit frequent, stable antibody docking with prolonged dwell times, whereas PD‐L1‐low luminal sEVs display predominantly transient interactions despite detectable PD‐L1 expression. Patient‐derived sEVs show intermediate binding dynamics, revealing functional heterogeneity that is not captured by bulk protein measurements. By integrating binding frequency and interaction dwell time, we establish a quantitative framework for nanoscale functional phenotyping of individual vesicles. These findings identify ligand accessibility as a distinct biophysical property complementary to protein abundance and establish HS‐AFM as a platform for investigating membrane protein function at single‐vesicle resolution.

Signal transduction is initiated when activated cell-surface receptors physically engage intracellular signaling proteins. Although structural and biochemical studies have established the molecular architecture of receptor tyrosine kinase (RTK) signaling, the earliest dynamic events that couple receptor activation to intracellular signal propagation have remained inaccessible because existing approaches provide static structural snapshots or ensemble-averaged measurements rather than direct observation of molecular motion. Here, using high-speed atomic force microscopy (HS-AFM), we directly visualize these initial molecular events at the single-molecule level in real time. Using fibroblast growth factor receptor 1 (FGFR1) as a model, we show that receptor autophosphorylation releases its intrinsically disordered juxtamembrane region, enabling extensive conformational dynamics of the kinase domain that permit productive engagement of the signaling adaptor FRS2α, a key initiator of downstream RTK signaling. In contrast, a kinase-inactive mutation or the clinically approved FGFR inhibitor futibatinib restricts receptor motion, stabilizes a compact receptor configuration, and markedly reduces adaptor engagement. Molecular dynamics simulations suggest that the disruption of inhibitory interactions between the juxtamembrane region and the kinase domain provides a structural explanation for the observed receptor dynamics. Together, our findings uncover a previously inaccessible physical mechanism underlying the initiation of RTK signaling and establish HS-AFM as a powerful platform for visualizing receptor dynamics, providing a framework for mechanistic studies of signal transduction and for the development and mechanistic evaluation of compounds that modulate receptor signaling.

Recurrence and metastasis remain major causes of cancer mortality, sustained by therapy–resistant micrometastatic cells. Bone is a frequent site of breast–cancer relapse, yet the cues that reawaken disseminated cells remain poorly defined. We identify a previously unrecognized, highly plastic CXCL16⁺ macrophage population that integrates tumor–associated macrophage programs found in distant metastatic sites such as lung and brain with non–tumor disease–associated traits in bone marrow. These CXCL16⁺ macrophages establish a transient niche that restrains disseminated cancer–cell proliferation. Single–cell transcriptomics delineate functional remodeling of myeloid niches within the bone metastatic microenvironment: a CXCL16⁺ macrophage niche that transiently constrains metastatic growth, and G–CSF⁺ macrophage and neutrophil niches that reignite tumor outgrowth. In primary tumors, cancer–associated fibroblasts (CAFs) aberrantly secrete G–CSF in response to cancer–cell signals, expanding G–CSF–receptor–positive subset of cancer cells with high metastatic potential. In advanced human bone metastases, CXCL16⁺ macrophages localize to CAF–rich stroma but are excluded from cancer–cell clusters, indicating immune evasion. Together, these findings uncover CAF–bone–marrow cross–talk as a therapeutic target linking stromal inflammation, immune remodeling, and metastatic progression.

One-carbon (1C) metabolism is frequently upregulated in cancer to support anabolic growth and nucleotide biosynthesis. However, the contribution of mitochondrial 1C metabolism to cancer stemness and metastatic progression remains incompletely understood. Here, we identify methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), a mitochondrial 1C metabolic enzyme, as a critical regulator of breast cancer stemness, tumor initiation, and lung metastasis. Genetic depletion of MTHFD1L in triple-negative breast cancer (TNBC) cell lines and patient-derived breast cancer models markedly impaired proliferation, sphere formation, tumorigenicity, and lung colonization. Metabolomic profiling revealed extensive metabolic rewiring following MTHFD1L loss, characterized by accumulation of the purine biosynthetic intermediates SAICAR and AICAR together with perturbations in glycolytic and pentose phosphate pathways. Importantly, suppression of lung metastasis was accompanied by reduced expression of the stemness-associated transcription factor SOX2 and decreased proliferative activity in metastatic lesions. Collectively, our findings establish MTHFD1L as a key metabolic dependency linking mitochondrial 1C metabolism to stem-like properties and metastatic progression in breast cancer, and highlight MTHFD1L as a promising therapeutic target in metastatic TNBC.

Approved KRAS inhibitors have shown limited therapeutic benefit over standard chemotherapy in PDAC and often encounter acquired resistance due to additional genetic alterations. RAS and RB1 functionally antagonize each other, which explains why RB1 is rarely mutated in KRAS-driven tumors. In PDAC cells, CDK4/6 inhibition induced cellular senescence accompanied by partial apoptosis. However, additional treatment with a senolytic agent or an ERK inhibitor promoted more efficient tumor cell elimination. While CDK4/6 inhibition downregulated KRAS activity, it concurrently upregulated EGFR signaling in a SASP and JNK-dependent manner. Deprivation of EGFR signaling after CDK4/6 inhibition triggered apoptosis in senescent cells in a manner similar to the treatment with a senolytic agent. In contrast, specific inhibition of KRAS induced modest enhancement of EGFR activity and SASP in a JNK-independent manner. Collectively, our study proposes that the CDK4/6 inhibitor may achieve greater therapeutic efficacy when combined with the EGFR inhibitor than KRAS inhibitor monotherapy.

Chemotherapy resistance remains a major obstacle for eradicating metastatic cancer cells in distant organs. We identified that endothelial cells (ECs) in the lungs, where breast cancer cells often metastasize, form a chemoresistant perivascular niche for disseminated breast cancer cells. By investigating the lung EC secretome activated by metastasis, we found that serine protease inhibitor family E member 1 (SERPINE1), encoded by Serpine1, is upregulated in metastasis‐associated lung ECs. This upregulation shields cancer cells from paclitaxel‐induced apoptosis and promotes cancer stem cell properties. Serpine1 expression appears to be driven by YAP‐TEAD activation in lung ECs that lose cell–cell contact, a phenomenon associated with increased vascular permeability in lungs affected by metastasis. Crucially, pharmacological inhibition of SERPINE1 enhances the chemotherapy sensitivity of metastatic breast cancer cells in the lung. Overall, our findings underscore the pivotal role of the vascular niche, which produces SERPINE1, in conferring chemoresistance to breast cancer cells during metastatic progression in the lungs.

Time-lapse scanning ion conductance microscopy visualizes subcellular nanoscale structural dynamics at the edges of breast cancer cells.

Triple-negative breast cancer (TNBC) lacks the expression of hormone and HER2 receptors and is highly malignant with no effective therapeutic targets. In TNBC, the cancer stem-like cell (CSC) population is considered to be the main cause of resistance to treatment. Thus, the therapeutic targeting of this population could substantially improve patient survival. Here, we identify the RNA-binding protein ZCCHC24 as enriched in the mesenchymal-like TNBC population. ZCCHC24 promotes the expression of a set of genes related to tumorigenicity and treatment resistance by directly binding to the cis -element “UGUWHWWA” in their mRNAs, thereby stabilizing them. One of the ZCCHC24 targets, ZEB1, is a transcription factor that promotes the expression of cancer stemness genes and reciprocally induces ZCCHC24 expression. ZCCHC24 knockdown by siRNAs shows a therapeutic effect and reduces the mesenchymal-like cell population in TNBC patient-derived xenografts. ZCCHC24 knockdown also has additive effects with the BET inhibitor JQ1 in suppressing tumor growth in TNBC patient-derived xenografts.

Air pollution, especially particulate matter (PM), is a significant environmental pollution worldwide. Studying the chemical, environmental, and life-related cellular physical characteristics of size-fractionated PMs is important because of their different degrees of harmful effects on human respiratory tracts and organ systems, causing severe diseases. This study evaluates the chemical components of size-fractionated PMs down to PM_0.1 collected during a biomass-burning episode, including elemental/organic carbon and trace elements. Single particle sizes and distributions of PM_0.1, PM_0.5-0.1, PM_1.0-0.5, and PM_2.5-1.0 were analyzed by scanning electron microscopy and Zeta sizer. Two commonly used cell lines, e.g., HeLa and Cos7 cells, and two respiratory-related cell lines including lung cancer/normal cells were utilized for cell cytotoxicity experiments, revealing the key effects of particle sizes and concentrations. A high-speed scanning ion conductance microscope explored particle-stimulated subcellular physical characteristics for all cell lines in dynamics, including surface roughness (SR) and elastic modulus (E). The statistical results of SR showed distinct features among different particle sizes and cell types while a E reduction was universally found. This work provides a comprehensive understanding of the chemical, environmental, and cellular physical characteristics of size-fractionated PMs and sheds light on the necessity of controlling small-sized PM exposures.

Chronic metabolic stress paradoxically elicits pro-tumorigenic signals that facilitate cancer stem cell (CSC) development. Therefore, elucidating the metabolic sensing and signaling mechanisms governing cancer cell stemness can provide insights into ameliorating cancer relapse and therapeutic resistance. Here, we provide convincing evidence that chronic metabolic stress triggered by hyaluronan production augments CSC-like traits and chemoresistance by partially impairing nucleotide sugar metabolism, dolichol lipid-linked oligosaccharide (LLO) biosynthesis and N-glycan assembly. Notably, preconditioning with either low-dose tunicamycin or 2-deoxy-D-glucose, which partially interferes with LLO biosynthesis, reproduced the promoting effects of hyaluronan production on CSCs. Multi-omics revealed characteristic changes in N-glycan profiles and Notch signaling activation in cancer cells exposed to mild glycometabolic stress. Restoration of N-glycan assembly with glucosamine and mannose supplementation and Notch signaling blockade attenuated CSC-like properties and further enhanced the therapeutic efficacy of cisplatin. Therefore, our findings uncover a novel mechanism by which tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.