Alpha-fetoprotein (AFP) is a classic biomarker for hepatocellular carcinoma (HCC). AFP-positive HCC (AFP+ HCC) has been intensively investigated; however, the genomic, transcriptomic and microenvironmental characteristics of AFP-negative HCC (AFP- HCC) remain to be deciphered. Here we show that tumors display mild differences in genetic alterations between AFP- HCC and AFP+ HCC patients, while AFP- HCC exhibits hyperactive arachidonic acid metabolism. Furthermore, the transcription activity of androgen receptor (AR) is significantly increased in AFP- HCC and plays a positive regulatory role in arachidonic acid metabolism and its metabolite 11,12-epoxyeicosatrienoic acid (11,12-EET). The tumor-derived 11,12-EET exhibits high affinity for EGFR that promotes the migration and tube formation of endothelial cells in vitro. Combination of lenvatinib and bicalutamide (an AR antagonist) enhances the therapeutic efficacy for AFP- HCC. Overall, we uncover the AR-mediated hyperactive arachidonic acid metabolism in AFP- HCC, and reveal AR-11,12-EET-EGFR axis-induced angiogenesis, providing a promising strategy of combined AR antagonist with lenvatinib for AFP- HCC treatment.
© 2025. The Author(s).

Biliary tract cancer (BTC) has a poor prognosis with limited treatment options. This phase 2 trial randomized 80 patients with unresectable/metastatic BTC 1:1 to sintilimab, anlotinib, and gemcitabine/cisplatin (SAGC) or chemotherapy alone (GC). At 13.4-month median follow-up, SAGC significantly improved median progression-free survival (8.5 vs. 6.3 months; HR 0.48, 95% CI 0.22-0.64, p = 0.005) and objective response rate (51.4% vs. 29.4%), with higher grade 3/4 adverse events (75.0% vs. 43.6%). Post hoc analysis showed enhanced efficacy with anlotinib 8 mg versus 10 mg (ORR 54.5% vs. 38.8%). In AKT/YAP tumor models, low-dose anlotinib (3 mg/kg) combined with sintilimab improved vascular perfusion, T-cell cytotoxicity, and cytokine secretion compared to high-dose (6 mg/kg). These findings demonstrate improved efficacy and manageable toxicity with SAGC, particularly at the 8 mg anlotinib dose, suggesting low-dose regimens may optimize antitumor response while mitigating adverse effects. Trial registration number ClinicalTrials.gov Identifier: NCT04300959.
© 2025. The Author(s).

Investigating proteases and protease inhibitors produced and secreted by primary cells is complicated by the presence of exogenous ones in cell culture medium containing fetal bovine serum. With this protocol, we generate human monocyte-derived macrophages in the absence of exogenous proteases and protease inhibitors by using a serum replacement for the final 24 h of macrophage culture. We describe steps for seeding and differentiating monocytes into macrophages. We then detail procedures for macrophage activation, culturing, and protein collection. For complete details on the use and execution of this protocol, please refer to Bernaerts et al.1.
Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.

CD1d-restricted invariant Natural Killer (iNK) T cells are a suitable candidate for allogeneic Chimeric Antigen Receptor (CAR) T cell therapy as they do not cause graft-versus-host disease (GvHD) due to the monomorphic nature of CD1d proteins. However, the phenotypic and functional heterogeneity of iNK T cells from adult donors (AD) may lead to the inconstant CAR-iNK T cell products. Cord blood-derived (CB) iNK T cells, in contrast, exhibit inter-donor homogeneity in phenotype including uniform CD4 expression and are enriched in memory iNK T cell populations. Thus, we evaluated the preclinical therapeutic potential of iNK T cells derived from cord blood (CB) as an off the shelf CAR T cell therapy platform, given the dominant presence of CD4+ iNK T cells. First, CB-derived iNK T cells were extremely enriched with CD4+ iNK T cells that express various NK receptors and display iNK-TCR mediated cytotoxicity but in a lesser degree than AD-derived CD4- iNK T cells. When engineered with an 8F4CAR targeting the acute myeloid leukemia-associated antigen PR1 presented in HLA-A2*01, CB-8F4CAR-iNK T cells showed a greater expansion capacity with higher CD62L expression than AD-8F4CAR-iNK T cells but with similar 8F4CAR expression and iNK T purity. CB-8F4CAR-iNK T cells displayed in vitro cytotoxicity against PR1/HLA-A2+ primary Acute Myeloid Leukemia (AML) and cell lines better than AD-8F4CAR iNK T cells and maintained potent cytotoxicity in repeated antigenic challenges. Moreover, CB-8F4CAR-iNK T cells showed anti-leukemia activity in vivo in a dose dependent manner. Lastly, CB-8F4CAR-iNK T cells were polarized to produce Th2-biased cytokines but in a lesser amount after 8F4CAR-mediated leukemia cytolysis compared to iNK-TCR mediated activation. In conclusion, consistent CD4+ phenotype, superior expansion capacity, and enhanced CD62L expression of CB-CAR-iNK T cells suggest that they may provide an alternative off-the-shelf source for effective CAR-iNK T cell therapy, while reducing the risk of severe cytokine release syndrome through their immunomodulatory properties. Thus, our results support the potential use of CB-iNK T cells as an allogeneic CAR-T cell therapy platform as they maintain a potent cytotoxicity with potentially better safety profile given a Th2-biased cytokine production upon activation.
Copyright © 2025 Grefe, Trujillo-Ocampo, Clinton, He, Yu, Li, Ma, Shpall, Molldrem and Im.

Systemic lupus erythematosus (SLE) is a typical autoimmune disease characterized by the overproduction of autoantibodies and type I interferon, which damages its own tissues, causing multiple organ damage. B cells are thought to play a major role in the pathogenesis of SLE. As a DNA methylation reader, Methyl-CpG-binding domain protein 2 (MBD2) has been extensively studied in the contexts of innate immunity, adaptive immunity, and autoimmune diseases. However, its specific role in B cells and SLE remains unexamined. Herein, we found that MBD2 was highly expressed in B cells of SLE patients and positively correlated with disease activity. Knockout of MBD2 in B cells disturbed B-cell differentiation, dampened B-cell activation, B-cell receptor (BCR) signaling, and T-cell-dependent humoral immune responses in mice. What's more, MBD2 deficiency effectively attenuated lupus-like symptoms, reduced the germinal center responses, and decreased anti-dsDNA antibodies in lupus model mice. Mechanistically, MBD2 selectively bound to the methylated CpG of Lef-1 induced by IFN-α, inhibiting the transcription and expression of Lef-1, which repressed Pten transcription and expression, thereby promoting PI3K-Akt-mTOR signaling. This study first demonstrated the role of MBD2 in the pathogenesis of SLE and provided a new target for SLE.
© 2025. The Author(s).