MSR1+ tumor-associated macrophages (TAMs) have been implicated in various malignancies; however, their functional role in Hepatocellular carcinoma (HCC) remains poorly defined. This research seeks to clarify the roles of MSR1+ TAMs in HCC and their influence on the tumor immune microenvironment. Clinical and experimental data indicate that high levels of MSR1+ TAMs correlate with poor prognosis in HCC patients. Transcriptomic analyses and in vitro as well as in vivo functional assays revealed that the immunosuppressive activity of MSR1+ TAMs is closely linked to their secretory profile. MSR1 enhances IL-6 secretion by activating the NF-κB signaling pathway, subsequently facilitating the recruitment of myeloid-derived suppressor cells (MDSCs). This cascade diminishes CD8+ T cell infiltration and effector function, promoting an immunosuppressive tumor microenvironment. In preclinical models, the simultaneous inhibition of MSR1 and PD-L1 markedly reduced tumor growth more effectively than either treatment alone. Our findings demonstrate that MSR1+ TAMs contribute to hepatocellular carcinogenesis through the NF-κB/IL-6 signaling axis by promoting MDSCs accumulation and impairing CD8+ T cell responses. Effectively targeting MSR1+ TAMs can overcome resistance to anti-PD-L1 therapy, offering a promising new immunotherapeutic approach for HCC.

Supplementary Figure S1 contains a schematic overview illustrating recurrent disease foci and sampling locations among participants enrolled in the study.

Supplementary Figure S7 illustrates macrophage phenotypic and functional alterations in peripheral blood and tumor tissues during immune rechallenge, including polarization and differentiation trajectories.

Background: Local drug delivery can increase antitumor exposure while limiting systemic toxicity, but chemotherapy-only local treatment may not fully control residual tumor growth in immunosuppressive tumor microenvironments. This study aimed to develop and preliminarily evaluate ffky-antiCD3, a CD3-recognition peptide-functionalized self-assembling peptide platform for intratumoral doxorubicin (DOX) delivery. Methods: The Nap aromatic group in a previous Nap-ffky scaffold was removed to improve aqueous dispersibility, and the CD3-recognition sequence AKMGEGGWGANDY was introduced to generate ffky-antiCD3. The peptide/formulation was characterized by reversed-phase high-performance liquid chromatography, mass spectrometry, TEM, circular dichroism spectroscopy, and a preliminary in vitro DOX release assay under tumor-mimicking acidic conditions. Antitumor efficacy, tumor histopathology, image-based CD3/CD8 semi-quantification, splenic IFN-γ levels, serum biochemistry, organ coefficients, and major-organ histology were assessed after repeated intratumoral treatment in B16-F10 melanoma-bearing C57BL/6 mice. Results: ffky-antiCD3 formed assemblies with a β-sheet-rich secondary structure. TEM observation further showed heterogeneous irregular/network-like supramolecular assemblies, and the preliminary release assay suggested slower apparent DOX release from ffky-antiCD3/DOX than from free DOX at pH 6.5. Among the tested groups, ffky-antiCD3/DOX produced the strongest short-term tumor-growth inhibition and the lowest endpoint tumor weight during the 10-day observation period. Ki67 staining decreased, and TUNEL signals increased after ffky-antiCD3/DOX treatment, supporting reduced proliferation and enhanced apoptosis-related damage. CD3/CD8 staining and exploratory splenic IFN-γ measurements indicated preliminary immune-related changes associated with ffky-antiCD3-containing formulations. Body weight, organ weights, serum biochemical markers, and major-organ H&E staining revealed no obvious short-term toxicity signals under the tested regimen. Conclusions: ffky-antiCD3/DOX represents a candidate local peptide-based chemo-immunomodulatory formulation. Its immune mechanism, release behavior, biodistribution, and long-term efficacy and safety require further validation before strong mechanistic or translational claims are made.

Supplementary Figure S6 details tumor-infiltrating T cell phenotypes, functional pathway enrichment, and TCR clonotype relationships between circulating and tumor-resident T cell populations.

Supplementary table S3 includes treatment-related adverse events in the total treated patients

Breast cancer metastasis claims the majority of breast cancer-related deaths. Anoikis resistance is a key prerequisite for CTCs survival and metastasis. Previous studies have demonstrated that Nicotinamide N-methyltransferase (NNMT) plays a crucial role in cancer metastasis and apoptosis resistance. However, whether NNMT participates in breast cancer CTCs anoikis remains unexplored. In this study, the upregulation of NNMT was observed in CTCs from breast cancer patients and mouse CTCs models. NNMT in detached breast cancer cells is induced by FAK-STAT3 axis and resists anoikis through FAO activation, promoting CTCs survival. Mechanistically, NNMT promotes the expression of CPT1A and CD36 by suppressing PP2A methylation to enhance FAO. Furthermore, NNMT-induced FAO accelerates ROS clearance by maintaining NADP+/NADPH balance. In vivo experiments show that NNMT-knockdown, NNMT inhibitors and FAO inhibitors can all reduce lung metastases formation, suggesting that targeting NNMT-FAO suppresses the metastatic potential of breast cancer. Our study revealed that the upregulation of NNMT is induced by FAK-STAT3 axis, which contributes to CTCs anoikis resistance in breast cancer by activating FAO. Targeting NNMT may provide new therapeutic targets for metastatic breast cancer.

Supplementary Figure S8 shows re-analysis of an external single-cell dataset validating immune remodeling patterns associated with response to PD-1 plus lenvatinib treatment.

Sepsis is a life-threatening and time-critical condition caused by a dysregulated host immune response to infection. Heparin-binding protein (HBP) has emerged as a promising biomarker for early sepsis diagnosis, as its concentration correlates closely with disease severity and the risk of progression to severe sepsis. Rapid and accurate detection of HBP can support timely intervention and potentially improve patient outcomes. However, reliable detection of HBP in serum remains challenging because of its low abundance (typically in the pg to ng mL-1 range), and the limited sensitivity and reproducibility of conventional analytical approaches. In this work, we report a paper-based biosensor (p-Biosen) for the rapid and ultrasensitive detection of HBP in human serum. The assay employs an electrochemical signal amplification strategy based on tetramethylbenzidine (TMB) and horseradish peroxidase (HRP). A capture antibody specific to HBP is immobilized on the test line of a lateral flow strip, where HBP binds sequentially with the capture antibody and an HRP-labeled detection antibody to form a sandwich-type immunocomplex. The HRP catalyzes the oxidation of TMB, resulting in the formation of an electroactive TMB-TMBox precipitate at the test line. A paper-based electrode is subsequently positioned directly over the test line to electrochemically quantify the deposited precipitate, generating an amplified current signal proportional to the HBP concentration. This proposed p-Biosen exhibits excellent analytical performance, achieving a low detection limit of 1.4 pg mL-1 and a wide linear range of 1.5-100 pg mL-1. In addition, the p-Biosen was integrated with a wireless potentiostat enabling portable, point-of-care HBP testing, offering a practical platform for rapid clinical decision-making in sepsis management.

The cell cycle regulator p16 is overexpressed in multiple cancers, and its elevated expression frequently correlates with aggressive tumor behavior and poor prognosis. However, the development of therapeutic agents directly targeting p16 has remained challenging. Antibody-drug conjugates (ADCs) represent an established modality for targeted drug delivery. To explore alternative formats that may address inherent challenges of conventional ADCs-such as potentially limited tumor penetration due to their large size and the risk of immunogenicity associated with the Fc domain-we engineered a novel p16-targeting antibody fragment-drug conjugate (AFDC). This conjugate is based on a humanized single-chain variable fragment (scFv) fused to a cell-penetrating peptide, aiming to achieve efficient intracellular delivery of the cytotoxic payload doxorubicin via p16-mediated targeting. We isolated five unique p16-specific scFvs from hybridoma cells. Following humanization, each was fused with a cell-penetrating peptide (CPP) S413 to construct scFv-p16-S413 variants. Following affinity assessment, scFv-p16-S413-04 was selected as the backbone and conjugated to doxorubicin via a chemical linker to generate the AFDC. In cellular assays, the resulting AFDC exhibited a p16 expression-dependent selectivity, showing stronger cytotoxicity against p16-high cancer cells (HeLa, BT-549) than against p16-low cells (MDA-MB-231, LO2, HEK-293 T). This selective activity correlated with target-dependent internalization. Furthermore, in p16-positive triple-negative breast cancer (TNBC) organoid models, AFDC treatment markedly reduced cell viability, underscoring its potent antitumor activity. Collectively, this proof-of-concept study supports the potential therapeutic value of a p16-targeted AFDC for p16-high tumors and illustrates the viability of the AFDC platform as an emerging strategy to target intracellular proteins.

Supplementary Figure S3 summarizes immune cell composition and proportional changes in peripheral blood, based on single-cell RNA sequencing across response groups and treatment time points.

Supplementary Figure S11 contains representative multiplex immunohistochemistry images illustrating tertiary lymphoid structures in tumor tissues.

Treatment options are limited for women with advanced endometrial cancer after platinum-based chemotherapy. In this single-arm, multicenter phase II CLEAR-EC trial with a safety run-in, we evaluated cadonilimab, a bispecific antibody targeting PD-1 and CTLA-4, plus lenvatinib as second-line or later therapy (ClinicalTrials.gov: NCT05824481). Three patients were enrolled in the safety run-in, followed by 29 additional patients after the recommended phase II dose was established. The primary endpoint was objective response rate; secondary endpoints included progression-free survival, overall survival, duration of response, disease control rate, and safety. Prespecified exploratory analyses assessed circulating tumor DNA in relation to efficacy, with additional post-hoc biomarker analyses. No dose-limiting toxicities occurred, establishing lenvatinib 16 mg once daily plus cadonilimab 10 mg/kg every three weeks as the recommended phase II dose. The trial met its prespecified primary endpoint, with an objective response rate of 40.6% (95% confidence interval, 23.7–59.4). Disease control rate was 81.3% (95% confidence interval, 63.6–92.8), median progression-free survival was 13.8 months, and median overall survival was not reached. Grade ≥3 treatment-related adverse events occurred in 40.6% of patients. Early on-treatment circulating tumor DNA levels were associated with progression-free and overall survival. Immune checkpoint blockade has emerged as a therapeutic option for patients with endometrial cancer. Here the authors report the results of a phase II trial of cadonilimab (a bispecific antibody targeting PD-1 and CTLA-4) combined with lenvatinib in advanced endometrial cancer.

Supplementary table S1 provides a summary of patient baseline clinical characteristics

Supplementary table S4 provides all the information of antibodies used in this study.

Supplementary Figure S13 provides a schematic summary of spatiotemporal genomic and immune determinants associated with response to immune rechallenge therapy.

Dairy cows during the transition period frequently experience negative energy balance (NEB), leading to elevated circulating non-esterified fatty acids (NEFA), which is a major risk factor for fatty liver disease. This study aimed to investigate the protective effects of epigallocatechin gallate (EGCG) on hepatocellular injury induced by NEFA, with an emphasis on elucidating the molecular pathways through which EGCG mitigates hepatic lipotoxicity. We observed that cows with fatty liver exhibited elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), and gamma-glutamyl transferase (GGT). Proteomic and Western blot analyses further revealed that the levels of cell apoptosis and endoplasmic reticulum (ER) stress in fatty liver dairy cows were significantly increased. Meanwhile, in palmitic acid (PA)-treated AML12 cells, EGCG pretreatment significantly alleviated hepatocyte apoptosis, as evidenced by a decreased Bax/Bcl2 ratio and a reduced apoptosis rate. Moreover, EGCG also mitigated ER stress by downregulating GRP78, HSP70, ATF4, and CHOP, and inhibited activation of the unfolded protein response (UPR), as indicated by reduced expression of XBP1, ATF6, phosphorylated IREα and PERK. Besides, in the ER stress cell model induced by tunicamycin (TM), it was further verified that EGCG can effectively alleviate hepatocyte apoptosis. The results indicate that EGCG alleviates the hepatocyte damage induced by NEFA through regulating the ER stress and UPR signaling, highlighting its potential as a natural intervention method for fatty liver diseases in transition dairy cows.

Background/Aims Liver transplantation (LT) following total hepatectomy is a life-saving treatment for hepatocellular carcinoma (HCC). The HCC recurrence after LT hinders the effectiveness of the procedure. The objective of this study is to develop a pre-operative risk stratification model based on a liquid biopsy. Methods We conducted a comprehensive multi-omics study of 260 HCC patients from three centers, including clinical data, low-coverage whole-genome sequencing of cell-free DNA (cfDNA) from plasma, as well as whole-exome, single-nucleus RNA, and spatial transcriptomics from matched tumor and non-tumor tissues. Results We identified cfDNA-derived copy number alteration (CNA) signatures associated with post-transplant recurrence. By integrating cfDNA-derived CNA profiles with single-cell transcriptomic data, we traced recurrence-associated cfDNA to a distinct subpopulation of malignant cells within the primary tumor. These cells were embedded in a pro-metastatic microenvironment of specialized endothelial subtypes and cancer-associated fibroblasts. Notably, most recurrence-associated lesions were detectable in cfDNA prior to liver transplantation (LT). Building on these insights, we developed the ZJU Criteria based on CNA fragments and tumor markers, a pre-LT risk prediction tool that integrates conventional clinical factors with cfDNA-derived CNA signatures, and validated it using internal and independent external cohorts. Conclusion Our findings suggest that post-transplant recurrence commonly originates from advanced subclones that emerge late during tumor evolution. The ZJU Criteria provides an accurate, non-invasive strategy that significantly improves pre-LT risk stratification and clinical decision-making for patients with HCC.

Immunotherapy has limited success in pancreatic ductal adenocarcinoma (PDAC) due to an immune exclusive tumor microenvironment (TME) that lacks many cytokines necessary for Natural Killer (NK) and T cell responses. Here, we design multiplexed mRNAs encoding interleukins, chemokines, and interferons as a safe and effective cytokine therapy for PDAC. Intratumoral injection of IL-12, IL-18, CCL5, CXCL10, and IFNβ mRNAs achieves robust yet transient cytokine expression, leading to NK and CD8+ T cell activation and reduced tumor growth and fibrosis in PDAC transplant mouse models. Combining cytokine with tumor antigen mRNAs enhances dendritic cell antigen presentation and CD8+ T cell priming locally and systemically that prolongs animal survival after a single dose. Remarkably, nanoparticle encapsulation of the cytokine/antigen mRNA cocktail allows systemic administration and local delivery to autochthonous PDAC tumors in mice, culminating in curative responses in 50% of animals and antigen-reactive T cell persistence. These results suggest that multiplexed mRNA approaches to deliver cytokines and antigens generally absent in the TME could pave the way for effective immunotherapy in PDAC.