Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload‐associated ZDHHC12 primarily participates in maintaining the activation of CD8+T cells in renal cell carcinoma. (a) UMAP plot showing the subpopulation classification of CD8+ T cells ( GSE121636 ). (b) UMAP visualization of ZDHHC12 expression across different CD8+ T cell subsets. (c) Violin plot depicting ZDHHC12 expression patterns in various CD8+ T cell subpopulations. (d) BALB/c mice fed either a high‐iron‐load diet or a normal diet were subcutaneously inoculated with Renca tumor cells on the dorsum, and starting from the day of inoculation, 200 µg of anti‐CD8 depletion antibody (αCD8) or an isotype control antibody was intraperitoneally administered every three days; tumor volume was measured from day 7 post‐inoculation. Data were presented as mean ± SD. e and f, Western blot (e) and RT‐qPCR (f) analyses were performed on CD8+ T cells sorted from renal cancer tissues of both the normal diet and high‐iron diet groups. (g–i), The figure illustrates schematic diagrams of different treatment groups (g). Specifically, tumor cells were isolated and sorted from the tumor tissues of the renal cancer model. Subsequently, the collected tumor cells were subcutaneously injected into the dorsum of either Zdhhc12 wild‑type or knockout mice. Tumor size was measured regularly, and growth curves were plotted (h). On day 19, the mice were sacrificed, and tumors were excised and weighed (i). (j–m), Portions of tumor tissues were processed for cell extraction, staining, and flow cytometry analysis to evaluate CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, the proportions of CD3+/CD45+ (j), CD8+/CD3+ (k), GZMB+/CD8+ (l), and IFN‐γ+/CD8+ (m) T cells within tumor masses were quantified. (n,o) Portions of tumor tissues were embedded, sectioned, stained, and subjected to immunofluorescence analysis to assess CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, multiplex staining was performed for DAPI/CD3/CD8/GZMB, and the proportions of positive cells were quantified (n); similarly, multiplex staining for DAPI/CD3/CD8/IFN‐γ was conducted with subsequent quantification of positive cell ratios (o).

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Activation Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation, Injection, Knock-Out, Extraction, Staining, Flow Cytometry, Cell Function Assay, Immunofluorescence, Multiplex Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload promotes the ubiquitin‐proteasome pathway degradation of ZDHHC12 by enhancing its binding with TRIM28. (a) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentrations for 48 h. (b) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentration gradients for 48 h. (c) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentrations for 48 h. (d) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentration gradients for 48 h. (e) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay after treatment with or without FAC (20 µg/mL) for 48 h. (f) HA‐ZDHHC12 was transfected into 293T cells, and co‐immunoprecipitation was performed using an anti‐HA‐ZDHHC12 antibody, followed by mass spectrometry to identify potential ZDHHC12‐interacting proteins. The peptide spectrum of TRIM28 is shown. (g) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay with or without TRIM28 knockout. (h) Schematic diagrams depicting the protein domain architecture of TRIM28 and the TRIM28 △RING truncation mutant. (i) Sorted CD8+ T cells were stably transfected with the plasmids illustrated in the figure, followed by cell collection for Western blot analysis and quantitative assessment. (j) After transfection with the indicated plasmids, 293T cells were treated with FAC, followed by MG132 treatment, and then collected for Co‐IP and Western blot analysis. (k) After treatment with FAC (0, 20, 40 µg/mL), 293T cells were treated with MG132, collected, and subjected to Co‐IP and Western blot analysis. (l) After transfection with the indicated plasmids for 24 h, 293T cells were collected for Co‐IP and Western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Concentration Assay, Transfection, Immunoprecipitation, Mass Spectrometry, Knock-Out, Mutagenesis, Stable Transfection, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12 inhibits cuproptosis in CD8+T cells through interaction with FDX1. (a) We reviewed the mass spectrometry results and found that FDX1 may interact with ZDHHC12, and the peptide spectrum of FDX1 is shown in the figure. (b) HA‐ZDHHC12 and Flag‐FDX1 were transfected into 293T cells, followed by cell lysis and co‐immunoprecipitation using Flag or HA antibodies, and then analyzed by western blot. (c) 293T cells were lysed and subjected to co‐immunoprecipitation with ZDHHC12 or FDX1 antibodies, followed by western blot detection. (d) Immunofluorescence staining was performed in 293T cells using ZDHHC12 and FDX1 antibodies, and images were acquired and analyzed by confocal microscopy. (e) GST pull‐down assay was performed using the recombinant protein of ZDHHC12. (f) GST pull‐down assay was performed using the recombinant protein of FDX1. (g) Protein structure prediction and molecular docking were conducted for ZDHHC12 and FDX1. (h,i) Western blot (h) and RT‐qPCR (i) were performed in 293T cells after transfection with the indicated plasmids. (j,k) Western blot (j) and RT‐qPCR (k) were performed in 293T cells after transfection with the indicated plasmids. (l) Western blot analysis was conducted in 293T cells 72 h after transfection with the indicated plasmids. (m) Western blot analysis was performed in 293T cells after transfection with the indicated plasmids. (n) CD8+ T cells were isolated from renal cancer tissues of Zdhhc12 WT and Zdhhc12 KO mice and subjected to western blot analysis. (o) After successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (p,q) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and immunostained with DLAT and Mito‐tracker, then imaged by confocal microscopy (p) to quantify (q) DLAT oligomerization. (r) After successful transfection with the indicated plasmids in 293T cells, cells were treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu for 48 h in the presence or absence of tetrathiomolybdate (TTM) (1 µ m ) as indicated, followed by CCK‐8 assay to measure and quantify cell viability. (s,t) Following successful transfection with the indicated plasmids in 293T cells, cells were collected after treatment with or without tetrathiomolybdate (TTM) (1 µ m ), then immunostained with DLAT and Mito‐tracker and imaged by confocal microscopy (s) to quantify (t) DLAT oligomerization. (u) After successful transfection with the indicated plasmids in CD8+ T cells, cells were treated with or without tetrathiomolybdate (TTM) (1 µ m ), then collected for western blot analysis. (v) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (w) After successful transfection with the indicated plasmids in sorted CD8+ T cells, cells were collected for western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Mass Spectrometry, Transfection, Lysis, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Pull Down Assay, Recombinant, Quantitative RT-PCR, Isolation, CCK-8 Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12‐mediated palmitoylation of FDX1 at Cys152/155 promotes its degradation. (a) In 293T and CD8+T cells, FDX1 was immunoprecipitated using an anti‐FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed either in the presence or absence of hydroxylamine (HAM) treatment, with subsequent streptavidin‐HRP pulldown of biotin‐conjugated proteins to specifically detect palmitoylated FDX1. (b) In 293T cells, FDX1 was immunoprecipitated using an anti‐ FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed with or without hydroxylamine (HAM) treatment, and subsequently enriched with streptavidin‐HRP to isolate biotin‐conjugated proteins, thereby enabling the detection of FDX1 palmitoylation levels in both 2‐BP (25 µ m , 24 h)‐treated and untreated conditions. (c) 293T cells treated with or without palmitic acid azide were collected for Click‐IT reaction and streptavidin pulldown. (d) 293T cells were treated with different concentrations of 2‐BP (+, 20 µ m ; ++, 40 µ m ) for 24 h, then collected for western blot analysis. (e) 293T cells treated with or without 2‐BP (25 µ m , 24 h) were exposed to CHX and collected at different time points for western blot analysis. (f) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (g) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (h) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (i) 293T cells transfected with the indicated plasmids were treated with or without 2‐BP (25 µ m , 24 h) after 24 h, then collected for western blot analysis. (j) The peptide spectrum for FDX1 palmitoylation site identification. (k) Conservation sequences of FDX1 C152 and FDX1 C155 sites across different species. (l) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment. m, AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (n) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment, followed by quantitative analysis of FDX1 palmitoylation levels. (o) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis. (p) CD8+ T cells were transfected with the indicated plasmids, then collected and stained with the indicated fluorescent dyes for flow cytometry analysis. (q) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Immunoprecipitation, Western Blot, Knock-Out, Transfection, Pull Down Assay, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Development of lipid nanoparticles encapsulating Zdhhc12 mRNA (Zdhhc12‐LNP). (a) Schematic diagram of the LNP synthesis process; (b) The average zeta potential and the average particle size; (c) The average PDI and the average encapsulation efficiency; (d) Representative cryo‐EM image showing the morphology of synthesized LNPs. (e) Spectrum showing the purity of the sample. (f) Representative chromatogram showing mRNA capping efficiency. (g) Western blot analysis of Zdhhc12 expression in CD8+ T cells isolated from mice and treated with control, blank LNP, or Zdhhc12‐encapsulated LNP, following a 24 h pre‐stimulation with 2 µg/mL anti‐CD3/CD28 and subsequent 24 h incubation with LNPs. (h) ABE assay and Western blot analysis were performed on CD8+ T cells isolated from mice to assess the palmitoylation level of Fdx1, where cells were first cultured for 24 h post‐infection without anti‐CD3/CD28, restimulated with 2 µg/mL anti‐CD3/CD28 for 24 h, incubated with LNPs for 24 h, and then harvested for analysis. (i) Renca cells were subcutaneously injected into the backs of BALB/c mice, LNPs were administered via intratumoral injection, and when tumors reached an appropriate size, tumors were harvested for sorting of CD8+ T cells, followed by Western blot analysis to assess Zdhhc12 expression level in CD8+ T cells. (j–m) Renca cells were subcutaneously injected into the dorsal region of BALB/c mice. After tumor establishment, mice received intratumoral injection of LNPs or no injection, and serum levels of ALT (j), AST (k), CRE (l), and BUN (m) were measured at appropriate time points. Ns, not significant. (n,o) BALB/c mice were subcutaneously injected with Renca cells, intratumorally administered Zdhhc12‐LNP or control, and intraperitoneally injected with 200 µg anti‐CD8 (αCD8) or control; tumor growth was recorded starting from day 7 post‐tumor inoculation (n), and flow cytometry analysis of CD8+ T cell content in the peripheral blood of mice (n = 5) was performed at the end of the experiment (o), with data presented as mean ± SD.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Zeta Potential Analyzer, Encapsulation, Cryo-EM Sample Prep, Synthesized, Western Blot, Expressing, Isolation, Control, Incubation, Cell Culture, Infection, Injection, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Under normal iron conditions, ZDHHC12‐mediated palmitoylation of FDX1 promotes FDX1 degradation by the mitochondrial matrix protease AFG3L2, ultimately suppressing cuproptosis in CD8+ T cells and maintaining their immune clearance function. Under iron overload conditions, increased binding between ZDHHC12 and TRIM28 leads to ZDHHC12 degradation, consequently inducing cuproptosis in CD8+ T cells and facilitating immune escape.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Binding Assay