Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Copper-bound metabolite of disulfiram augments NIS activity and radioiodide u ptake. (A) Overview of rational design and reformulation drug strategies used in study. (B) RAI uptake of copper gluconate [Cu(II)]-treated 8505C–NIS and TPC-1-NIS cells alone or in combination with disulfiram (DSF) versus untreated (UT). (C) Schematic illustrating metabolic conversion of DSF to copper diethyldithiocarbamate [Cu(DDC) 2 ]. (D) RAI uptake of Cu(DDC) 2 -treated 8505C–NIS and TPC-1-NIS cells. (E) Same as (D) but using parental 8505C and TPC-1 cells. (F) Western blot analysis of NIS expression in Cu(DDC) 2 -treated 8505C–NIS and TPC-1-NIS cells; HE, higher exposure. (G and H) Confocal imaging of 8505C–NIS-HA (G) and TPC-1-NIS-HA (H) cells treated with Cu(DDC) 2 or vehicle (DMSO). Confocal images represent HA expression (green), NIS expression (red), and a merged image (yellow). Arrows (white) indicate regions of greater NIS plasma membrane localisation; HA, haemagglutinin. Scale bar: 20 μm. (I) Western blot analysis of NIS protein levels at the PM relative to Na+/K + ATPase following the cell-surface biotinylation assay (CSBA) in 8505C–NIS cells after Cu(DDC) 2 treatment. Control: Biotin tag omitted ( upper ), total protein before biotin separation ( lower ). (J) RAI uptake of Cu(DDC) 2 -treated human primary thyrocytes. Data presented as mean ± SEM (n = 3); one-way ANOVA, Dunnett's or Tukey's post hoc test (ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001); unpaired two-tailed t-test ( # P < 0.05).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, Western Blot, Expressing, Imaging, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Control, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Dual agonist effect of Cu(DDC) 2 enhances NIS activity . (A – C) RAI uptake in parental 8505C cells (A), parental SW1736 cells (B) and human primary thyrocytes (C) following PAX8-siRNA depletion and Cu(DDC) 2 treatment. CON: scrambled control siRNA. (D) Relative NIS mRNA in human primary thyrocytes following PAX8-siRNA depletion and Cu(DDC) 2 treatment. (E) Relative PAX8 mRNA in human primary thyrocytes following PAX8-siRNA depletion. (F) RAI uptake in 8505C–NIS and TPC-1-NIS cells following NPL4-or VCP-siRNA depletion and Cu(DDC) 2 treatment. (G) Western blot analysis of NIS, NPL4 and VCP in 8505C–NIS and TPC-1-NIS cells after NPL4-or VCP-siRNA depletion. (H) Same as (F) but in human primary thyrocytes. (I) NanoBiT evaluation of protein: protein interaction between NIS and VCP in living HeLa cells treated with CB5339 or Cu(DDC) 2 versus controls. (J) Schematic illustrating NanoBRET assay to monitor proximity of NIS with plasma membrane protein KRAS, as well as subcellular markers RAB5 (early endosome) and RAB11 (recycling endosome). Created with BioRender.com . Modified from Read ML et al. Clinical Cancer Research, 2024. (K and L) NanoBRET evaluation of NIS localisation at the PM (K, KRAS) or in ER-golgi (L, RAB1) in live HeLa and HEK293 cells treated with Cu(DDC) 2 . Data presented as mean ± SEM (n ≥ 3), one-way ANOVA, Dunnett's or Tukey's post hoc test (ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001), unpaired two-tailed t-test ( # P < 0.05, ## P < 0.01, ### P < 0.001).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, Control, Western Blot, Clinical Proteomics, Membrane, Modification, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Copper-bound metabolite stimulates NIS activity to enhance radionuclide uptake in vivo . (A) Schematic illustrating albumin nanoencapsulation of Cu(DDC) 2 . Lower : RAI uptake in 8505C–NIS cells treated with Cu(DDC) 2 in DMSO or albumin nanoencapsulated (ALB). (B) Overview of in vivo study to investigate the effect of Cu(DDC) 2 given by IP (step 1) or IV routes (step 2) on thyroidal NIS function in WT BALB/c mice. (C and D) 99m TcO4 - uptake (C, n = 6–11) and relative NIS mRNA (D) in thyroid glands from Cu(DDC) 2 -ALB treated WT BALB/c mice given by IP route. Total animals used = 18). (E and F) Same as (C and D) but Cu(DDC) 2 -ALB given by IV route at indicated doses (n = 5–7). Total animals used = 21. ( G ) Pearson correlation analysis between thyroidal 99m TcO4 - uptake (FC, log 2 ) and relative NIS mRNA (FC, log 2 ) in Cu(DDC) 2 -ALB treated WT BALB/c mice as outlined (B). 95% CI (upper/lower) are shown. (H and I) Relative PAX8 and NKX2-1 mRNA in thyroids from Cu(DDC) 2 -ALB treated WT BALB/c mice given by IP (H) or IV (I) routes. (J and K) Same as (H and I) but relative TPO and TG mRNA. (L) Schematic illustrating the dual impact of Cu(DDC) 2 on NIS function to enhance RAI uptake by inducing NIS mRNA and inhibiting VCP activity. Inset —promoter/enhancer regions of TPO , TG and NIS genes with relative positions of NKX2-1 and PAX8 binding sites. (M) Representative H&E stained images of thyroid tissue from Tg-rtTA/tetO-BRAF V600E mice fed with DOX ( upper ) versus normal ( lower ) chow for 7 days. Scale bars, 100 μM. (N) 99m TcO4 - uptake in thyroid tissue in DOX chow fed Tg-rtTA/tetO-BRAF V600E mice treated with Cu(DDC) 2 -ALB given by IV route (n = 3–6). Total animals used = 9. Data presented as mean ± SEM, unpaired two-tailed t-test (ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). Images created with BioRender.com .

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, In Vivo, Binding Assay, Staining, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Transcription factor and VCP/proteostasis genes predict recurrence risk in RAI-treated PTC . (A) Volcano plot comparing log 2 FC with q -value (-log base 10) for the THCA PTC cohort and 337 transcription factor (TF) genes. (B) Volcano plot comparing log 2 FC with q -value (-log base 10) for the GSE33630 dataset and 323 TF genes. (C) Venn diagram illustrating TF genes common to both GSE33630 and THCA PTC datasets. (D) Volcano plot illustrating log 2 FC compared to q -value (-log base 10) for disease-free survival (DFS) in the BRAF-like, RAI-treated PTC cohort and 337 TF gene panel. (E) Mean number of dysregulated TF genes stratified into high-risk group (bars; left y-axis) and recurrence rate (white crosses; right y-axis) in patient clusters 1 to 4 (n = 20–49). (F) Representative Kaplan–Meier analysis of DFS for BRAF-like, RAI treated PTC stratified into patient clusters 1 to 4, log-rank test. (G) Box and whisker plot showing NIS expression (log 2 ) in BRAF-like, RAI-treated PTC stratified into patient clusters 1 to 4, Kruskal–Wallis test followed by Dunn's post hoc test (ns, not significant, ∗∗∗ P < 0.001). (H) LASSO regression analysis used to construct a 22 TF gene riskscore classifier. LASSO coefficient plot (loglambda). Y-axis: coefficient value; x-axis (lower): log(λ) value, and x-axis (upper): gene number. (I – K) ROC analysis (I) and Kaplan–Meier curve of the 22 TF gene riskscore classifier in BRAF-like, RAI-treated PTC (J) or RAI-treated PTC (K). (L) Kaplan–Meier analysis of DFS in BRAF-like, RAI-treated PTC ( left ) or RAI-treated PTC ( right ) stratified with the dual TF + VCP riskscore classifier. (M) Uni- ( left ) and multivariate analysis ( right ) of RAI-treated PTC (n = 211) stratified with the dual TF + VCP classifier, adjusting for the covariates age, sex, disease stage and ATA risk group in the multivariate model. (N) Same as (M) but with the entire TCGA THCA cohort (n = 399).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Whisker Assay, Expressing, Construct