Journal: Materials Today Bio

Article Title: Metformin glycyrrhetinic acid binary injectable hydrogel for synergistic tumor immunotherapy via spatiotemporal microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102749

Figure Lengend Snippet: In vitro reprogramming of the tumor microenvironment (TME) by Fe 3 O 4 NPs@Met-GA-H. ( A, B ) Immunofluorescence analysis and quantitative analysis of HMGB1 exposure (A, red) and CRT exposure (B, red) in B16-F10 cells following different treatments. Blue: DAPI (n = 3). ( C ) Immunofluorescence staining of CD86,iNOS and CD206 in macrophages (n = 3). ( D ) Immunofluorescence staining of PD-L1 (n = 3). ( E-G )Quantitative analysis of CD86,iNOS and CD206 expression. ( H ) Quantitative analysis of PD-L1 expression (n = 3). Data are presented as Mean ± SD. Pairwise comparisons against the control group were performed using Student's t-test; differences among multiple groups were assessed by one-way ANOVA. Ns: not significant (P > 0.05); ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus Ctrl. # P < 0.05, ## P < 0.01, ### P < 0.001 versus the IL-4 group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alexa Fluor® 594 AffiniPure Goat Anti-Rabbit IgG (H + L) (Cat. No. 111-585-003, dilution: 1:250) was sourced from Jackson ImmunoResearch Inc. Rabbit anti-CD86 antibody (Cat. No. WL05184, dilution: 1:200), rabbit anti-iNOS antibody (Cat. No. WL0992a, dilution: 1:200), rabbit anti-CD206 antibody (Cat. No. WL06177, dilution: 1:200), and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (H + L) (Cat. No. WLA032, dilution: 1:200) were purchased from Wanlei Bio-Tech Co., Ltd. Rabbit anti-PD-L1 antibody (Cat. No. bs-1103R, dilution: 1:250) was obtained from Beijing Bioss Biotechnology Co., Ltd. PerCP/Cyanine5.5 anti-mouse CD45 Recombinant Antibody (Cat. No. 157612, dilution: 1:200), PE anti-mouse CD11c Antibody (Cat. No. 117308, dilution: 1:200), APC anti-mouse CD86 Antibody (Cat. No. 159216, dilution: 1:200), FITC anti-mouse CD80 Antibody (Cat. No. 104706, dilution: 1:200), PE/Cyanine7 anti-mouse I-A b Antibody (Cat. No. 116420, dilution: 1:200), Zombie R718TM Fixable Viability Kit (Cat. No. 423116, dilution: 1:200), FITC anti-mouse CD3 Antibody (Cat. No. 100204, dilution: 1:100), PE anti-mouse CD4 Antibody (Cat. No. 100408, dilution: 1:200), APC/Cyanine7 anti-mouse CD8a Antibody (Cat. No. 100714, dilution: 1:200), APC anti-mouse/human CD11b Antibody (Cat. No. 101212, dilution: 1:200), APC/FireTM 750 anti-mouse F4/80 Antibody (Cat. No. 123152, dilution: 1:200), PE/DazzleTM 594 anti-mouse CD206 (MMR) Antibody (Cat. No. 141732, dilution: 1:200), and PE/Cyanine7 anti-mouse CD86 Antibody (Cat. No. 159208, dilution: 1:200) were purchased from BioLegend.

Techniques: In Vitro, Immunofluorescence, Staining, Expressing, Control

Journal: Materials Today Bio

Article Title: Metformin glycyrrhetinic acid binary injectable hydrogel for synergistic tumor immunotherapy via spatiotemporal microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102749

Figure Lengend Snippet: In vivo immune activation of Fe 3 O 4 NPs@Met-GA-H. ( A, B ) Immunofluorescence staining of HMGB1 (A, red) and CRT (B, red) in tumor tissues following various treatments. Blue: DAPI; scale bars: 100 μm. (n = 5). ( C, D ) Immunofluorescent staining of CD206 (C, green) and CD86 (D, green) in tumor sections. Blue: DAPI; scale bars: 100 μm. (n = 5). ( E ) Immunofluorescence staining of PD-L1 expression (red) in tumors across treatment groups. Blue: DAPI; scale bars: 100 μm. (n = 5). ( F, G ) Flow cytometry analysis of MHC-II expression (F) and CD80 + +CD86 + populations (G) in DCs from spleens. (n = 5). ( H ) Immunofluorescence co-staining of CD4 (green) and CD8 (red) in tumor tissues. Blue: DAPI; scale bars: 100 μm. (n = 5). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. Ns: not significant ( P > 0.05); ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the Ctrl. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alexa Fluor® 594 AffiniPure Goat Anti-Rabbit IgG (H + L) (Cat. No. 111-585-003, dilution: 1:250) was sourced from Jackson ImmunoResearch Inc. Rabbit anti-CD86 antibody (Cat. No. WL05184, dilution: 1:200), rabbit anti-iNOS antibody (Cat. No. WL0992a, dilution: 1:200), rabbit anti-CD206 antibody (Cat. No. WL06177, dilution: 1:200), and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (H + L) (Cat. No. WLA032, dilution: 1:200) were purchased from Wanlei Bio-Tech Co., Ltd. Rabbit anti-PD-L1 antibody (Cat. No. bs-1103R, dilution: 1:250) was obtained from Beijing Bioss Biotechnology Co., Ltd. PerCP/Cyanine5.5 anti-mouse CD45 Recombinant Antibody (Cat. No. 157612, dilution: 1:200), PE anti-mouse CD11c Antibody (Cat. No. 117308, dilution: 1:200), APC anti-mouse CD86 Antibody (Cat. No. 159216, dilution: 1:200), FITC anti-mouse CD80 Antibody (Cat. No. 104706, dilution: 1:200), PE/Cyanine7 anti-mouse I-A b Antibody (Cat. No. 116420, dilution: 1:200), Zombie R718TM Fixable Viability Kit (Cat. No. 423116, dilution: 1:200), FITC anti-mouse CD3 Antibody (Cat. No. 100204, dilution: 1:100), PE anti-mouse CD4 Antibody (Cat. No. 100408, dilution: 1:200), APC/Cyanine7 anti-mouse CD8a Antibody (Cat. No. 100714, dilution: 1:200), APC anti-mouse/human CD11b Antibody (Cat. No. 101212, dilution: 1:200), APC/FireTM 750 anti-mouse F4/80 Antibody (Cat. No. 123152, dilution: 1:200), PE/DazzleTM 594 anti-mouse CD206 (MMR) Antibody (Cat. No. 141732, dilution: 1:200), and PE/Cyanine7 anti-mouse CD86 Antibody (Cat. No. 159208, dilution: 1:200) were purchased from BioLegend.

Techniques: In Vivo, Activation Assay, Immunofluorescence, Staining, Expressing, Flow Cytometry

Journal: Materials Today Bio

Article Title: Ultrasound-activated miR-195-5p/shikonin nanobubbles remodel immunosuppressive microenvironment via immunogenic cell death to potentiate PD-1/PD-L1 blockade in hepatocellular carcinoma

doi: 10.1016/j.mtbio.2025.102216

Figure Lengend Snippet: US-responsive lipid NBs co-delivering miR-195-5p and shikonin (miR-195-5p/SK-NBs) were engineered to synergize PD-L1 suppression and ICD for enhanced antitumor immunity. The NBs achieved tumor-targeted accumulation via passive/active mechanisms, ensuring biosafety. US-triggered microbubble destruction enabled localized miR-195-5p release, downregulating PD-L1 expression, functionally mimicking PD-1/PD-L1 blockade. Concurrently, NB-mediated SK delivery overcame systemic toxicity, inducing robust ICD with the secretion of CRT, HMGB1, and ATP. In HCC models, miR-195-5p/SK-NBs reprogrammed the TIME, amplifying splenic lymphocyte proliferation, cytotoxic CD8 + T cell infiltration, IFN-γ production, proportion and cytotoxic activity of CTL activity, improving the tumor suppression of anti–PD-1 antibody. Created with BioRender.com .

Article Snippet: Membranes were blocked with 5 % non-fat milk in TBST for 1 h at room temperature, followed by overnight incubation at 4 °C with rabbit anti-PD-L1 polyclonal antibody (1:500 dilution, Wanleibio, Shenyang, China).

Techniques: Expressing, Activity Assay

Journal: Materials Today Bio

Article Title: Ultrasound-activated miR-195-5p/shikonin nanobubbles remodel immunosuppressive microenvironment via immunogenic cell death to potentiate PD-1/PD-L1 blockade in hepatocellular carcinoma

doi: 10.1016/j.mtbio.2025.102216

Figure Lengend Snippet: miR-195-5p-mediated suppression of PD-L1 expression. ( A) Bioinformatic prediction of the miR-195-5p/PD-L1 targeting interaction. (B) Schematic diagram of the pMIR-REPORTER empty vector. (C) Construction strategy of the luciferase reporter plasmid containing the PD-L1 3′-UTR. (D) Sequencing validation of the PD-L1 3′-UTR luciferase reporter plasmid. (E–F) Luciferase reporter assay for miR-195-5p activity in Hepa 1–6 cells, which were co-transfected with miR-195-5p mimics, miR mimic NC, miR-195-5p inhibitor, or miR inhibitor NC for 48 h. Then Hepa 1–6 cells, which were co-transfected with pcDNA 3.1(+), miR-195-5p, miR mimic NC, miR-195-5p mimic, miR inhibitor NC, and miR-195-5p inhibitor for 48h. (G) Quantitative analysis of PD-L1 expression levels across groups by Western blot from panel H. (H) Representative Western blot bands of PD-L1. (I) Statistical analysis of PD-L1 expression from panel J. (J) Flow cytometry plots of PD-L1 expression in Hepa 1–6 cells 48 h post-transfection.

Article Snippet: Membranes were blocked with 5 % non-fat milk in TBST for 1 h at room temperature, followed by overnight incubation at 4 °C with rabbit anti-PD-L1 polyclonal antibody (1:500 dilution, Wanleibio, Shenyang, China).

Techniques: Expressing, Plasmid Preparation, Luciferase, Sequencing, Biomarker Discovery, Reporter Assay, Activity Assay, Transfection, Western Blot, Flow Cytometry

Journal: Materials Today Bio

Article Title: Ultrasound-activated miR-195-5p/shikonin nanobubbles remodel immunosuppressive microenvironment via immunogenic cell death to potentiate PD-1/PD-L1 blockade in hepatocellular carcinoma

doi: 10.1016/j.mtbio.2025.102216

Figure Lengend Snippet: miR-195-5p/SK-NBs-mediated suppression of PD-L1 expression in vitro . Hepa 1–6 and HepG2 cells were treated with N-NBs + US, miR-195-5p-Lipo3000, and miR-195-5p-NBs + US for 48 h. (A) Flow cytometry histograms of surface PD-L1 expression. (B–C) Quantitative analysis of PD-L1 expression levels from panel C in (B) HepG2 cells and (C) Hepa 1–6 cells. (D–E) Quantification of PD-L1 protein levels from panel F in (D) HepG2 cells and (E) Hepa 1–6 cells. (F)Western blot of total PD-L1 protein expression.

Article Snippet: Membranes were blocked with 5 % non-fat milk in TBST for 1 h at room temperature, followed by overnight incubation at 4 °C with rabbit anti-PD-L1 polyclonal antibody (1:500 dilution, Wanleibio, Shenyang, China).

Techniques: Expressing, In Vitro, Flow Cytometry, Western Blot

Journal: Materials Today Bio

Article Title: Ultrasound-activated miR-195-5p/shikonin nanobubbles remodel immunosuppressive microenvironment via immunogenic cell death to potentiate PD-1/PD-L1 blockade in hepatocellular carcinoma

doi: 10.1016/j.mtbio.2025.102216

Figure Lengend Snippet: In vivo biodistribution and tumor analysis of ICD and PD-L1 protein (A) Ex vivo fluorescence imaging of Dil and Dil-miR-195-5p/SK-NBs at 24 h post-injection. H22 xenograft–bearing mice were randomized into Control, N-NBs + US, free SK, miR-195-5p-NBs + US, SK-NBs + US, and miR-195-5p/SK-NBs + US. Mice were treated on days 1, 4, 7, 10, and 13. Then, tumors were harvested on 14 d for the following assays: (B) HMGB1 in tumor tissues. (C) Immunofluorescence staining of CRT in tumor tissues (scale bar = 100 μm). (D) Quantification of CRT fluorescence intensity from panel C. (E) Immunofluorescence staining of PD-L1 in tumor tissues (scale bar = 100 μm). (F) Quantitative analysis of PD-L1 fluorescence intensity from panel E.

Article Snippet: Membranes were blocked with 5 % non-fat milk in TBST for 1 h at room temperature, followed by overnight incubation at 4 °C with rabbit anti-PD-L1 polyclonal antibody (1:500 dilution, Wanleibio, Shenyang, China).

Techniques: In Vivo, Ex Vivo, Fluorescence, Imaging, Injection, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Tumor intrinsic regulation of PD-L1 and of interferon Type I via an SLC25A1-driven mitochondrial pathway, influences the anti-tumor immune response

doi: 10.1101/2025.09.20.677512

Figure Lengend Snippet: A. Schematic representation of the experiments. B-D. PD-L1 and SLC25A1 protein levels in control and SLC25A1 expressing H1299 spheres (B), or in spheres treated with CTPI-2 (C), or transduced with the SLC25A1 shRNA (D). E. PD-L1 and SLC25A1 protein levels in the indicated tumor (H1975 and HCC1937) and patient-derived (T5) NSLCs untreated or treated with CTPI-2. F. PD-L1 and SLC25A1 protein levels in HCC827 cells treated with conditioned culture supernatant that were derived from SLC25A1 low or high SLC25A1 spheres.

Article Snippet: Immunohistochemical staining of mouse PD-L1 was performed using anti-mouse PD-L1 Rabbit polyclonal antibody (ABclonal, A1645).

Techniques: Control, Expressing, Transduction, shRNA, Derivative Assay

Journal: bioRxiv

Article Title: Tumor intrinsic regulation of PD-L1 and of interferon Type I via an SLC25A1-driven mitochondrial pathway, influences the anti-tumor immune response

doi: 10.1101/2025.09.20.677512

Figure Lengend Snippet: A. PD-L1 protein levels in control or EtBr-treated H1299 spheres used at two different concentrations (indicated by +). B. PD-L1 protein levels in SLC25A1-H1299 spheres infected with control lentivirus (pLKO) or with pLKO lentivirus harboring a specific shRNA for IFNAR1. C-D . The knock-down of the cGAS or STAT1 does not affect PD-L1 mRNA (C) or protein (D) levels in SLC25A1 expressing cells. E . Treatment of naïve H1299 with fumarate (DMF), itaconate, succinate and malate for 16 hours and immuno-blot with the indicated proteins. F-G. Keap1, PD-L1 and SLC25A1 protein levels in cells over-expressing SLC25A1 (F) or harboring the SLC25A1 shRNA (G). Note that the PD-L1 blot in panel F is the same as in . H-I . Keap1, PD-L1 and SLC25A1 protein levels in H1299 in spheres harboring control or the Keap1 shRNA (H), or, viceversa , over-expressing Keap1 (I, high), versus control cells.

Article Snippet: Immunohistochemical staining of mouse PD-L1 was performed using anti-mouse PD-L1 Rabbit polyclonal antibody (ABclonal, A1645).

Techniques: Control, Infection, shRNA, Knockdown, Expressing

Journal: bioRxiv

Article Title: Tumor intrinsic regulation of PD-L1 and of interferon Type I via an SLC25A1-driven mitochondrial pathway, influences the anti-tumor immune response

doi: 10.1101/2025.09.20.677512

Figure Lengend Snippet: A. Tumor volumes for the indicated treatment groups from two independent experiments. B. Tumor growth curves of control and SLC25A1 over-expressing B16F0 tumors treated with either PD-L1 (αPD-L1) or isotype control antibodies. C. The abundance of the indicated cell populations were analyzed by FACS. The error bars represent the standard deviation, asterisks refer to *p ≤ 0.05; **p ≤ 0.01 ; ** *p ≤ 0.001 by unpaired T -test. D. q-RT-PCR of the indicated macrophage markers. E. Summary of the results and proposed model (see also text for explanation).

Article Snippet: Immunohistochemical staining of mouse PD-L1 was performed using anti-mouse PD-L1 Rabbit polyclonal antibody (ABclonal, A1645).

Techniques: Control, Expressing, Standard Deviation, Reverse Transcription Polymerase Chain Reaction