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CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety–specific conjugation with deferoxamine–maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects. [ABSTRACT FROM AUTHOR]

The South Korean aquaculture industry has incurred considerable production losses due to various infectious diseases. Artificially synthesized polyinosinic–polycytidylic acid (poly I:C), structurally similar to double-stranded RNA (dsRNA) and cytidine–phosphate–guanosine oligodeoxynucleotides, can enhance immune responses and protect against diseases. Here, we investigated dsRNA molecules with different cytidine–phosphate–guanosine (CpG) motifs (dsRNA-CpGMix) as fused agents to treat Miamiensis avidus and viral hemorrhagic septicemia virus (VHSV) infection in olive flounders. We further investigated the efficacy of specific sequence motifs in dsRNA in modulating immunostimulatory effects. Fish treated with poly I:C or dsRNA-CpGMix exhibited higher survival rates than the control group. Olive flounder leukocytes stimulated with poly I:C or dsRNA-CpGMix showed increased scuticocidal activity in the presence of inactivated immune sera. dsRNA with CpG motif sequences induced higher resistance against M. avidus and VHSV infections than dsRNA without CpG motif sequences, and the dsRNA-CpGMix group showed upregulated ISG15 or Mx compared to the dsRNA-GFP group. Thus, dsRNA containing CpG motifs can be used as effective immunostimulants to enhance resistance against viral and parasitic diseases in olive flounder. The specific sequences of the CpG motifs in dsRNA may be important for enhancing immune responses and resistance against M. avidus and VHSV infections in olive flounders. [ABSTRACT FROM AUTHOR]

Liver cancer stem cells (LCSCs) contribute to the initiation, metastasis, treatment resistance, and recurrence of hepatocellular carcinoma (HCC). Therefore, exploring potential anticancer agents targeting LCSCs may offer new therapeutic options to overcome HCC treatment failure. Hovenia dulcis Thunberg (HDT), a tree from the buckthorn family found in Asia, exhibits various biological activities, including antifatigue, antidiabetic, neuroprotective, hepatoprotective, and antitumor activities. However, the therapeutic effect of HDT in eliminating LCSCs remains to be confirmed. In this study, we evaluated the inhibitory activity of ethanol, chloroform, and ethyl acetate extracts from HDT branches on the growth of Huh7-derived LCSCs. The ethyl acetate extract of HDT (EAHDT) exhibited the most potent inhibitory activity against the growth of Huh7 LCSCs among the three HDT extracts. EAHDT suppressed the in vitro self-renewal ability of Huh7 LCSCs and reduced tumor growth in vivo using the Huh7 LCSC-transplanted chick embryo chorioallantoic membrane model. Furthermore, EAHDT not only arrested the cell cycle in the G0/G1 phase but also induced receptor-interacting protein kinase 3/mixed-lineage kinase domain-like protein-mediated necroptosis and caspase-dependent apoptosis in Huh7 LCSCs in a concentration-dependent manner. Furthermore, the growth inhibitory effect of EAHDT on Huh7 LCSCs was associated with the downregulation of c-MET-mediated downstream signaling pathways and key cancer stemness markers. Based on these findings, we propose that EAHDT can be used as a new natural drug candidate to prevent and treat HCC by eradicating LCSCs. [ABSTRACT FROM AUTHOR]