Journal: Science Advances

Article Title: An in situ forming immunomodulatory hydrogel reinvigorates exhausted CD8 + T cells via preserving their mitochondrial fitness

doi: 10.1126/sciadv.adz0039

Figure Lengend Snippet: ( A ) Bio-TEM images of untreated 4T1 cells and 4T1 cells treated with TPGS for 4, 8 and 12 hours (h). Highlighted section indicate mitochondria (green), lysosomes (blue), mitochondrial-containing autophagosomes (red), autolysosome-like degradative compartments (yellow) and degraded mitochondrial (purple). Scale bar, 1 μm. ( B and D ) Fluorescence microscopy images (B) illustrating mitochondrial ROS levels in 4T1 cells after treating with different concentrations of TPGS, along with quantitative analysis (D) of mean fluorescence intensity (MFI) ( n = 3). Scale bar, 50 μm. ( C and E ) Representative flow cytometry plots (C) and quantitative analyses (E) of mitochondrial membrane potential depolarization via JC-1 staining in 4T1 cells after indicated treatments. ( F ) CLSM images of the colocalization of TPGS with mitochondria in 4T1 cells after 4 hours of incubation (Pearson’s coefficient, 0.825). Scale bar, 10 μm. ( G ) Western blot analysis and corresponding quantification of LC3-I/LC3-II and Parkin protein levels in 4T1 cells after indicated treatments ( n = 3). ( H ) CLSM images of immunofluorescence staining and corresponding quantitative analysis of BNIP3 protein expression in 4T1 cells after indicated treatments. Scale bar, 60 μm. ( I ) Illustration of target sites of conventional ICD inducers compared to TPGS. ( J ) CLSM images of ecto-CRT exposed on surface of 4T1 cells at 12 hours posttreatment with various ICD inducers. Scale bar, 40 μm. ( K ) Quantitative fluorescence intensity analysis of ecto-CRT ( n = 4) and detection of HMGB1 and ATP levels released into the cell supernatants by ELISA kit after 4T1 cells were treated with various ICD inducers ( n = 3). Data are presented as means ± SD. Statistical analyses [(D), (E), (G), (H), and (K)] were conducted using one-way ANOVA, followed by Tukey’s multiple-comparisons test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Reactive Oxygen Specimen Assay Kit, Mitochondrial Membrane Potential Assay Kit, and CCK8 Assay Kit were purchased from Beyotime Biotechnology Ltd.

Techniques: Fluorescence, Microscopy, Analysis, Flow Cytometry, Membrane, Staining, Incubation, Western Blot, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: An in situ forming immunomodulatory hydrogel reinvigorates exhausted CD8 + T cells via preserving their mitochondrial fitness

doi: 10.1126/sciadv.adz0039

Figure Lengend Snippet: ( A ) FESEM images showing TNTs between 4T1 cells and CD8 + T cells under untreated conditions or following TPGS treatment, respectively. Scale bars, 15 μm. ( B ) Quantitative analysis showing the distribution of lengths and widths of TNTs connecting the 4T1 cells and CD8 + T cells, as measured from the FESEM images ( n = 6). ( C ) CLSM images of TNT (yellow arrows) formation between cocultured 4T1 cells and CD8 + T cells, with mitochondrial hijacking events (red arrows). Scale bars, 10 μm. ( D ) Experimental design to quantify mitochondrial hijacking by 4T1 cells from immune cells. ( E ) Representative flow cytometry plots show the transfer of MitoTracker-labeled mitochondria from CD8 + T cells to CellTrace-labeled 4T1 cells after different treatments. ( F to I ) Oxygen consumption rates (OCRs) of FACS-sorted 4T1 cells (F) and CD8 + T cells (H) after coculture, with or without TPGS treatment. Monocultured 4T1 cells and CD8 + T cells served as controls. Basal respiration and spare respiratory capacity (SRC) of 4T1 cells (G) and CD8 + T cells (I) ( n = 3). ( J ) CLSM images of TPGS-mediated RHOT1 degradation improving mitochondrial mass in CD8 + T cells. Scale bars, 20 μm. ( K ) Western blot and corresponding analysis of RHOT1 expression in 4T1 cells after treatment with different ICD inducers ( n = 3). ( L ) Schematic illustration showing the all-in-one bioactivity of TPGS in inducing ICD in cancer cells and inhibiting mitochondrial hijacking via activation of the prodeath mitophagy pathway. Data are presented as means ± SD. Statistical analyses [(B), (G), (I), and (K)] were conducted using one-way ANOVA, followed by Tukey’s multiple-comparisons test. (D) and (L) were created in BioRender. qi, Y. (2026) https://BioRender.com/bg6yxhd .

Article Snippet: Reactive Oxygen Specimen Assay Kit, Mitochondrial Membrane Potential Assay Kit, and CCK8 Assay Kit were purchased from Beyotime Biotechnology Ltd.

Techniques: Analysis, Flow Cytometry, Labeling, Western Blot, Expressing, Activation Assay