Although antibody-drug conjugates (ADCs) have made substantial progress as targeted therapies, the range of suitable ADC payloads remains limited. In this study, the highly N-methylated cyclodepsipeptide [MeAla3-MeAla6]-coibamide (CA) was selected as a novel toxin for ADC construction due to its potent toxicity and unique mechanism of action. Using a quaternary ammonium salt approach, two linker-payload variants, MC-VA-PAB-CA and MC-GGFG-PAB-CA, with distinct cathepsin B (CTB)-cleavable linkers, were synthesized and assessed. Among them, MC-GGFG-PAB-CA demonstrated higher enzyme-responsive cleavage efficiency and superior plasma stability and was selected for conjugation with the epidermal growth factor receptor (EGFR) antibody cetuximab (Ctx), resulting in the formation of Ctx-CA. This conjugate exhibited EGFR-dependent antitumor activity, a pronounced "bystander killing effect", and a significant tumor suppression effect in mouse models. Furthermore, the applicability of this conjugation strategy was confirmed through validation with the HER2 antibody. These findings suggest that CA is a promising weapon for next-generation ADCs.

Tumor senescence is a critical mechanism underlying tumor progression and recurrence. A better understanding of how pre-metastatic circulating tumor cells (CTCs) exploit senescence to survive in the blood stream could help reveal vulnerabilities for therapeutic intervention. Using patient-derived melanoma CTC lines and xenograft models, we identified a role for the cytoskeletal regulator cortactin in mTOR/p53-dependent senescence. Cortactin localized to Rab7-positive endosomes and maintained late-endosomal homeostasis. Depletion of cortactin induced aberrant endosomal aggregates with mTOR accumulation and hyperactivation, subsequently leading to p53 activation, G0/G1 arrest, and cellular senescence. This oncogene-induced senescence (OIS) was characterized by induction of the senescence-associated secretory phenotype and β-galactosidase (SA-β-gal), loss of Ki-67 and lamin B1, and elevated mitochondrial reactive oxygen species (mtROS). Notably, a positive feedback loop between p53 and mtROS was essential for maintaining stable senescence in CTCs. Clinically, the proportion of SA-β-gal-positive senescent CTCs was significantly correlated with therapeutic resistance and disease progression in a prospective cohort of melanoma patients. A sequential strategy using cortactin depletion followed by an anti-Bcl-xL senolytic eliminated the persistent CTCs and suppressed blood-borne metastasis. Thus, this study uncovered a unique senescent CTC subpopulation regulated by a cortactin/mTOR/p53/mtROS axis that can be targeted to suppress metastatic progression of melanoma.

Supplementary Table S3, primer, shRNA and siRNA sequences

Supplementary figure S10. KD CTTN leads to aberrant Rab7-positive late endosomal aggregates with mTOR co-localization and activation

Supplementary Table S1, patient blood samples infomation

Supplementary Table S2, antibody infomation

Supplementary figure S2. CTTN KD in Mel-182-1 and PEM22 CTCs with BRAF mut/NRAS mut did not induce robust senescence

Supplementary figure S11. p53 is sparsely localized on Rab7 positive endosomal aggregates of Mel-167 cells

[This corrects the article DOI: 10.1371/journal.pone.0055839.].

Cell rounding during mitosis necessitates adaptive remodeling of plasma membrane and cortical cytoskeleton. However, the underlying mechanisms remain poorly elucidated. Here, we have identified Numb phosphorylation as a pivotal mechanism in the membrane-cytoskeleton remodeling associated with mitotic cell rounding. Upon mitotic entry, Aurora A phosphorylates Numb, leading to the dissociation of Numb from plasma membrane. This is crucial for proper plasma membrane retraction, since overexpression of a non-phosphorylatable mutant or a constitutively membrane-bound variant of Numb dramatically disrupts mitotic plasma membrane retraction. Mechanistically, releasing Numb from the plasma membrane enhances the myosin I-mediated membrane-to-cortex adhesion, thereby facilitating the plasma membrane retraction accompanied with cytoskeletal withdrawal. Further analysis showed that compromised plasma membrane retraction confines mitotic cell rounding and consequently leads to spindle orientation defects. Thus, our study elucidates a phosphorylation-mediated mechanism underlying plasma membrane retraction and underscores the functional importance of this process in the context of mitotic cell rounding.

Background: Diet-derived advanced glycation end products (dAGEs) are closely associated with obesity and metabolic disorders. This study investigates the therapeutic potential of myriocin (Myr), a sphingolipid synthesis inhibitor, in counteracting dAGE-induced obesity and its underlying mechanisms. Methods: Male C57BL/6J wild-type mice were randomly assigned to receive either a low-AGE diet or a high-AGE diet with or without the administration of myriocin for a duration of 24 weeks. At the end of the experimental period, blood samples, whole livers, and adipose tissues were harvested for subsequent biochemical, histological, and molecular analyses. Results: Using a 24-week high-AGE diet mouse model, we demonstrate that Myr significantly reduces body weight gain (by 76%) and adipose tissue accumulation, while alleviating hepatic steatosis. Myr improves glucose homeostasis by lowering fasting blood glucose (a 44.5% reduction), enhancing oral glucose tolerance, and restoring hepatic glycolysis/gluconeogenesis balance via upregulating glucokinase and suppressing G6pc. Notably, Myr reduces serum LDL-C, TG, and TC levels by 52.3%, 51.8%, and 48.8%, respectively, and ameliorates liver dysfunction as evidenced by normalized ALT/AST activities. Metabolomics reveal Myr reshapes amino acid, carbohydrate, and lipid metabolism pathways. Mechanistically, Myr suppresses lipogenesis by downregulating Srebp1, Fasn, and Acc, while activating AMPK-PGC1α signaling to enhance mitochondrial biogenesis (a 2.1-fold increase in mtDNA) and thermogenesis via Ucp1 upregulation in brown and white adipose tissues. Conclusions: Our findings unveil Myr as a novel dual regulator of lipid and glucose metabolism through AMPK-PGC1α-mediated mitochondrial activation, providing the first evidence of sphingolipid inhibition as a therapeutic strategy against dAGE-induced metabolic syndrome. This study establishes a multifaceted mechanism involving hepatic lipid regulation, adipose browning, and systemic metabolic reprogramming, advancing potential clinical applications for obesity-related disorders.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive form of cancer, characterized by limited treatment options and a poor prognosis. Immunological therapy is an emerging and promising strategy that has the potential to enhance treatment outcomes and extend the survival of patients with ICC. The role of CD4+ T cells in the development of cancer has attracted attention in previous years. However, the complexities of the tumor microenvironment (TME) impede the full understanding of the roles of CD4+ T cells in cancer. The present study used single-cell RNA sequencing to explore the heterogeneity of the TME during the development of ICC. The results demonstrated that CD4+ T cells were enriched in the TME of ICC and the ratio of regulatory T cells (CD4-forkhead box P3) to central memory T cells (CD4-interleukin 7 receptor) was markedly increased. Secreted phosphoprotein 1 (SPP1) and CD44 showed increased expression levels in tumor cells and T cells from ICC tumor tissues, respectively. Additionally, SPP1 gene expression levels were higher and the ratio of regulatory T cells to central memory T cells was increased in the late stage of ICC compared with the early stage. Elevated levels of SPP1 were associated with a poor prognosis for patients with ICC. Finally, analysis of cell-cell interactions, utilizing established receptor-ligand pairs, demonstrated that ICC tumor cells may engage with immune cells through an SPP1-CD44 axis. Therefore, the results suggest that ICC tumor cells impact CD4+ T-cell differentiation, which could alter the immune TME in ICC and potentially promote ICC tumor progression.

Plasma membrane proteins at cell surface are critical for numerous physiological and pathological processes and are primary targets for clinical drugs. Given that clustering of plasma membrane proteins by endogenous stimuli or pharmaceutical interventions serves as a key trigger for their internalization and degradation, this process critically influences their function and turnover. Inspired by this natural process, we developed a modular, protein-of-interest (POI) targeting degradation strategy by using a bifunctional chimera molecule composed of a POI-binding ligand and a self-assembling peptide (WIII/YIII). We term this strategy SAILTAC (Self-Assembling Peptide Induced Lysosomal Targeting Chimera) and demonstrate that these chimeras could efficiently degrade membrane-anchored GFP and the therapeutically relevant immune checkpoint PD-L1. An optimized dimeric chimera (YIII-BMS)₂ potently reduced PD-L1 across multiple cancer cell lines through the lysosomal pathway. Collectively, the SAILTAC strategy offers a versatile and targeted approach to degrade plasma membrane proteins, providing a new tool for nanomedicine application.

Infection with the hepatitis B virus (HBV) is a key risk factor for hepatocellular carcinoma (HCC) development and progression. It is widely recognized that immunopathological mechanisms are pivotal in the development of HBV-associated HCC; nevertheless, the specific underlying mechanisms through which HBV-induced modifications within the tumor microenvironment (TME) contribute to HCC pathogenesis have yet to be fully elucidated. In the present study, single-cell RNA sequencing was utilized to analyze and compare the immune landscapes between HBV-positive and HBV-negative HCC. These experiments revealed that HBV infection significantly modifies the composition and state of immune cells, leading to the suppression and exhaustion of T cells within the TME. Specifically, increases in the proportions of SLC4A10+ CD8+ T cells and IFITM3+ macrophages were observed, along with an upregulation of the gene SLC35F1 in various immune cell subtypes. Taken together, these findings have offered valuable insights into the alteration of the immunological microenvironment in HCC that is associated with HBV infection, suggesting possible targets for immunotherapeutic intervention.

Background:Sodium-glucose transporter 2 inhibitors (SGLT-2Is) and Semaglutide may increase the risk of sarcopenia and bone fragility in vulnerable populations, yet their effects on body composition in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) remain unclear. This study evaluated changes in body composition by SGLT-2Is alone or combined with Semaglutide. Methods:This retrospective cohort included T2DM-CKD patients treated with SGLT-2Is ± Semaglutide for ≥6 months. Body composition (fat, muscle, water, bone mineral content [BMC]) was measured via bioelectrical impedance analysis pre- and post-treatment. Results:Among 73 participants (SGLT-2Is: n = 61; combination: n = 12), both groups showed reductions in total fat mass, total muscle mass, total body water, and BMC. Combination therapy exhibited greater fat mass loss (-0.9 kg [IQR: -3.7,0.4] vs -0.6 kg [-1.7,0.7]; P = 0.011) and muscle mass decline (-1.1 ± 1.2 kg vs -0.4 ± 0.8 kg; P = 0.015) versus monotherapy. Fat mass index (FMI: -1.3 ± 2.4 kg/m² vs -0.2 ± 0.8 kg/m²; P = 0.008) and skeletal muscle index (SMI: -0.4 ± 0.3 kg/m² vs -0.2 ± 0.2 kg/m²; P = 0.002) reduction were also larger with combination therapy. However, muscle mass-to-body weight percentage was increased more in the combination group (1.2 ± 2.4% vs 0.2 ± 1.2%; P = 0.041). No differences between to groups in BMC, fat percentage, or fat-to-muscle ratio (P>0.05). Within the SGLT-2Is group, higher baseline SMI correlated with greater muscle loss, while higher baseline FMI was associated with attenuated BMC decline. Conclusion:SGLT-2Is with/without Semaglutide reduced body composition parameters of fat, muscle, water, and BMC in T2DM-CKD. Combination therapy exacerbated absolute muscle loss but increased the muscle mass-to-body weight percentage, without significantly altering fat-to-muscle ratio. Baseline muscle and fat mass may influence treatment-related changes. Long-term studies in high-risk populations are needed.

Cell surface receptor-targeted protein degraders hold promise for drug discovery. However, their application is restricted because of the complexity of creating bifunctional degraders and the reliance on specific lysosome-shuttling receptors or E3 ubiquitin ligases. To address these limitations, we developed an autophagy-based plasma membrane protein degradation platform, which we term AUTABs (autophagy-inducing antibodies). Through covalent conjugation with polyethylenimine (PEI), the engineered antibodies acquire the capacity to degrade target receptors through autophagy. The degradation activities of AUTABs are self-sufficient, without necessitating the participation of lysosome-shuttling receptors or E3 ubiquitin ligases. The broad applicability of this platform was then illustrated by targeting various clinically important receptors. Notably, combining specific primary antibodies with a PEI-tagged secondary nanobody also demonstrated effective degradation of target receptors. Thus, our study outlines a strategy for directing plasma membrane proteins for autophagic degradation, which possesses desirable attributes such as ease of generation, independence from cell type and broad applicability. Cheng et al. developed an autophagy-based targeted protein degradation platform by conjugating polyethylenimine to antibodies, designated as autophagy-inducing antibodies, which can degrade proteins in vivo and enable the degradation of multiple proteins at the same time.

Metastatic dormancy often refers to the stable cell cycle arrest of disseminated tumor cells (DTCs) at distant sites. However, whether circulating tumor cells (CTCs) in the blood microenvironment can enter a dormant state prior to extravasation and becoming DTCs remains unclear. Using patient-derived melanoma CTC lines and animal explant models (CDX), we identified a previously unrecognized role of the cytoskeletal regulator cortactin (encoded by CTTN) in controlling mTOR/p53-dependent senescence and metastatic dormancy. Cortactin was localized to Rab7-postive endosomes and engaged in late endosomal tethering and homeostasis. The depletion of cortactin resulted in the accumulation of aberrantly enlarged late endosomal aggregates that were positive for Rab7 and mTOR. The mTOR protein complex was accumulated and activated within these abnormal vesicular structures, leading to robust p53 activation through phosphorylation at serine (S) 15 and S33 sites. Consequently, melanoma CTCs underwent G0/G1 cell cycle arrest and entered cellular senescence. This unusual oncogene-induced senescence (OIS) mechanism was characterized by SASP upregulation, beta-galactosidase activity, depletion of Ki-67 and Lamin B1, and elevated mitochondrial ROS (mtROS) levels. Notably, a positive feedback loop between p53 and mtROS was essential for maintaining stable senescence in CTCs. In preclinical CDX mouse models, we developed a sequential therapeutic strategy combining cortactin depletion with anti-Bcl-xL senolytic drugs. Such "One-two punch" treatment strategy effectively eliminated viable CTCs and suppressed metastatic tumor growth in vivo. Thus, targeting cortactin to induce CTC senescence, followed by senolytic therapy, may represent a promising strategy to block CTC-mediated metastatic progression. ### Competing Interest Statement The authors have declared no competing interest.

Targeted degradation of membrane-associated proteins, which constitute a crucial class of drug targets implicated in diverse disease pathologies, has garnered considerable attention in chemical biology and drug discovery recently. Taking advantage of the endosomal entrapment of cell-penetrating peptides (CPPs) in delivering bioactive macromolecules, we successfully construct a CPP-based platform for specific degradation of cell surface proteins by conjugation of target protein-binding small molecules (SMs) with different CPPs, resulting in the formation of CPP-mediated lysosome-targeting chimeras (CPPTACs). Through the endo-lysosomal pathway, CPPTACs exhibit a remarkable ability to degrade clinically significant plasma membrane proteins, including PD-L1, CAIX, and CB2R. In contrast to LYTACs and similar technologies, CPPTACs drive the degradation of targets in a manner independent of specific lysosome-shuttling receptors, thus providing a widely applicable strategy for plasma membrane protein degradation, regardless of the cell types. Additionally, simpler structural design and broader therapeutic window for CPPTACs are expected since CPPs-mediated endocytosis and lysosomal degradation do not necessitate the three-component binding model typically required by other heterobifunctional degraders. Overall, consisting of small molecules and biocompatible cell-penetrating peptides, CPPTACs developed in this study represent a simple, adaptable, and effective approach for selectively degrading cell surface proteins in various cellular contexts with potential for application in both biological research and therapeutic interventions.

INTRODUCTION:The quality of traditional Chinese medicine (TCM) is a prerequisite for clinical efficacy. However, the existing quality evaluation methods are not strongly correlated with efficacy, and they are unable to adequately reflect the quality grade of Changii Radix (CR). OBJECTIVES:In this study, a biology-related chemical indicator quality grading prediction model was developed to predict the quality grade of CR. MATERIALS AND METHODS:Firstly, the quality grade of CR was pre-classified based on immunological activity. Subsequently, one-way analysis of variance, gray correlation analysis, and Pearson correlation analysis were employed to identify the chemical indicators associated with immunological activity. Finally, separately using chemical indicators as independent variables and quality grades as dependent variables, the logistic regression model and a multi-index weighted quality comprehensive evaluation index (QCEI) were constructed to predict the quality grade of CR. RESULTS:The results indicated that 27 batches of CR samples could be divided into three grades of I, II, and III. The gray correlation degrees and Pearson correlation coefficients between water-soluble extractives, polysaccharide, amino acid, and immunological activity all exceeded 0.8 and 0.4 (p < 0.05), respectively. Additionally, both the logistic regression model and QCEI could effectively predict the quality grade of CR, with the logistic regression model showing superior performance. CONCLUSION:This study is the first to establish a chemistry-biology integrated strategy for evaluating the quality grade of CR, providing a novel insight into the assessment of TCM quality grade.