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INTRODUCTION: Triple-negative breast cancer (TNBC) is characterized as the most unfavorable prognosis of the breast cancer subtype. Chemotherapy is currently the primary treatment owing to a persistent lack of effective alternative medicine. To fulfill this unaddressed clinical requirement, we utilized the PGLU-Fc-III-4C MsAb platform to develop a nanobioconjugate trispecific antibody (TsAb; CD3¡ÁCD137¡ÁCD276) that targeted CD3, CD137, and CD276, aiming to restrict the growth of TNBC tumors (4T1 model) and provide a novel therapeutic strategy. METHODS: The capacity for binding to target cells and anti-tumor effects of TsAb in vitro were evaluated. In vivo antitumor efficacy and biosafety were further assessed in the 4T1 subcutaneous tumor model. Anti-tumor immune responses induced by TsAb on tumor-infiltrating CD8+ T cells were monitored. RESULTS: The TsAb effectively bound and facilitated interactions between 4T1 tumor cells and T cells, significantly boosting the anti-tumor effect. TsAb promoted the expansion of tumor and splenic T lymphocytes and facilitated the recruitment of splenic and blood T lymphocytes to tumor tissues. Compared with the PGLU-Fc-III-4C-IgG treatment group, the TsAb treatment group had varying degrees of increase in CD8+ tissue-resident memory T cells (TRM), central memory T cells (TCM), and terminal effector memory T cells (TEM) in tumor tissues. The TsAb treatment group exhibited a significant increase of PD-1+ CD8+ T cells and TCF1-Tim-3+ CD8+ terminally exhausted T cells in tumor tissues. The safety profile demonstrated no obvious systemic toxicity. DISCUSSION: Briefly, TsAb mediated CD8+ T cell activation, proliferation, and terminal differentiation, accompanied by increasing cytokine production to eliminate the tumor. Meanwhile, no obvious systemic toxicity was observed. In general, the CD3¡ÁCD137¡ÁCD276 nanobioconjugate trispecific antibody provides a promising immunotherapeutic approach via regulating CD8+ T cell immune response for the treatment of triple-negative breast cancer.

Osthol (OST), a natural coumarin, exhibits anti-inflammatory and metabolism-regulating potential. This study investigated whether OST ameliorates obesity-associated metabolic dysregulation and inflammation by targeting ADRA1D-mediated T helper 17 (Th17) differentiation. High-fat diet (HFD)-induced obese mice were treated with OST. Metabolic parameters including body/organ weights, serum lipids, hepatic enzymes, and histopathology were assessed. Th17-related and inflammatory markers were evaluated via flow cytometry, ELISA, RT-qPCR, and Western blot. In vitro Th17 differentiation (primary murine CD4? T cells) and lipid metabolism (3T3-L1 adipocytes) models were used. ADRA1D was identified as a key target via bioinformatics and validated through overexpression in cells and mice. OST significantly reduced HFD-induced weight gain, liver and fat mass, serum triglycerides (TG), free fatty acids (FFA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic lipid deposition, and adipocyte hypertrophy. OST suppressed Th17 differentiation, CD4?IL-17A? and CD4?ROR¦Ãt? cell proportions, and pro-inflammatory cytokines (IL-17A, IL-6, TNF-¦Á), while elevating anti-inflammatory cytokines (IL-10, TGF-¦Â). OST downregulated IL-17RA, TRAF6, and Act1 expression and inhibited ERK1/2 and PI3K phosphorylation. In vitro studies confirmed the dose-dependent inhibitory effect of OST on Th17 polarization. Mechanistically, OST modulated Th17-related signaling via ADRA1D. ADRA1D overexpression partially reversed OST-mediated suppression of Th17 differentiation, expression of lipogenic genes (FASN, PPAR¦Ã), and lipid droplet accumulation. In vivo, ADRA1D overexpression attenuated the beneficial effects of OST on metabolic parameters and tissue inflammation, confirming ADRA1D dependence. OST ameliorates obesity-related metabolic dysregulation and inflammation by inhibiting ADRA1D-mediated Th17 differentiation, highlighting ADRA1D as a key mediator and potential therapeutic target for immunometabolic disorders.

Rapid and uniform labeling of plasma membrane proteins is essential for high-resolution imaging of dynamic membrane topologies and intercellular communication in live mammalian cells. Existing strategies for labeling live cell membranes, such as fluorescent fusion proteins, enzyme-mediated tags, metabolic bioorthogonal labeling, and lipophilic dyes, face trade-offs in the requirement of genetic manipulation, the presence of non-uniform labeling, the need for extensive preparation times, and limited choices of fluorophores. Here, we present a streamlined protocol that leverages N-hydroxysuccinimide (NHS)-ester chemistry to achieve rapid (¡Ü5 min), covalent conjugation of synthetic small-molecule dyes to surface-exposed primary amines, enabling pan-membrane-protein labeling. This workflow covers dye stock preparation, labeling for suspension and adherent cells, multiplex live-cell imaging, fusion protein co-staining (including insulin-triggered receptor endocytosis), 3D membrane visualization, and in vivo assays for visualizing membrane-derived material transfers between donor and recipient cells using a lymphoma T-cell mouse model. This high-density labeling approach is compatible with various cell types across diverse imaging platforms. Its speed, versatility, and stability make it a broadly applicable tool for studying plasma membrane dynamics and intercellular membrane trafficking. Key features ? Rapid high-density membrane labeling with small-molecule fluorescent dyes.? Enables live-cell multiplexed imaging, amenable to primary cells and cells expressing fluorescent fusion proteins, and supports in vivo studies of membrane-associated cell¨Ccell communications.? Compatible with various fluorescence imaging modalities.