Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Timely administration of drug combination improves chemoimmunotherapy of an immune-cold tumor

doi: 10.1016/j.jconrel.2025.02.075

Figure Lengend Snippet: (a) Schematic depiction of 2E’/PTX/CDN assembly. (b) TEM image of 2E’/PTX/CDN (5:1:0.1, w/w/w). Scale bar: 100 nm. (c) In vitro PTX release kinetics from 2E’/PTX/CDN in PBS containing 0.2% Tween-80 (n = 3 replicates of a representative batch, mean ± SD). (d) Confocal images of bone marrow-derived dendritic cells (BMDCs) incubated with free CDN or 2E’/PTX/CDN (5:1:0.1, w/w/w) at a concentration equivalent to 0.1 µg/ml FITC-labeled CDN for 8 h. Cell membrane was stained with Wheat Germ Agglutinin Alexa Fluor 633 Conjugate. Red: cell membrane; green: CDN. Scale bar: 50 µm. (e) Fluorescence intensity of BMDCs incubated with PBS, free CDN, or 2E’/PTX/CDN (5:1:0.1, w/w/w) at a concentration of 0.1 µg/ml FITC-labeled CDN for 8 h, measured by flow cytometry. n=3 replicates of a representative batch. (f) TLR5 activation indicated by SEAP production from HEK-Blue ™ mTLR5 reporter cells incubated with 2E’ or 2E’/PTX/CDN (5:1:0.1, w/w/w) for 24 h. Flagellin (Fla, 100 ng/mL), a known TLR5 agonist, was used as a positive control, shown in blue. n=3 replicates in a representative experiment. (g) B16F10 cells undergoing apoptosis and necrosis, stained with Annexin V-PI. The cells were treated with PBS (negative control) or 2E’/PTX/CDN at 4 μg/mL 2E’, 0.8 μg/mL PTX, and 0.08 μg/mL CDN for 24 h. The fractions of cell population undergoing apoptosis and necrosis were determined by dual-color analysis; n = 3 replicates in a representative experiment. (h) Calreticulin (CRT) exposure and ATP secretion of B16F10 melanoma cells after 24 h incubation with 2E’ (1.25 µg/mL), PTX (0.25 µg/mL), 2E’/PTX (1.25 µg/mL 2E’; 0.25 µg/mL PTX), 2E’/siPD-L1 (1.25 µg/mL 2E’; 1.125 µg/mL siPD-L1), or 2E’/PTX/CDN (1.25 µg/mL 2E’; 0.25 µg/mL PTX; 0.025 µg/mL CDN). CRT exposure was measured by flow cytometry. See Fig. S5 for gating strategy. n=3 replicates in a representative experiment. (i) Maturation of BMDCs incubated with PBS, 2E’ (5 µg/mL), PTX (1 µg/mL), CDN (0.1 µg/mL), 2E’/PTX (5 µg/mL 2E’; 1 µg/mL PTX), 2E’/CDN (5 µg/mL 2E’; 0.1 µg/mL CDN), or 2E’/PTX/CDN (5 µg/mL 2E’; 1 µg/mL PTX; 0.1 µg/mL CDN) for 24 h. n=3 replicates of a representative batch. (j) Left: Secretion of TNF-α and IFN-β by BMDCs incubated with PBS, 2E’ (5 µg/mL), CDN (0.1 µg/mL), or 2E’/PTX/CDN (5 µg/mL 2E’; 1 µg/mL PTX; 0.1 µg/mL CDN); right: TNF-α secretion by bone marrow-derived macrophages (BMDMs) incubated with free CDN or 2E’/PTX/CDN at 0.05 µg/mL CDN equivalent (CDN L and 2E’/PTX/CDN L ) or 0.1 µg/mL CDN equivalent (CDN H and 2E’/PTX/CDN H ). For both 2E’/PTX/CDN L and 2E’/PTX/CDN H , the weight ratio of 2E’:PTX:CDN was 5:1:0.1. n=3 replicates of a representative batch. (k) Cytotoxicity of 2E’/PTX/CDN to immune cells (BMDC and BMDM) and tumor cells (4T1 breast cancer cells, CT26 colon cancer cells, and B16F10 melanoma cells). The weight ratio of 2E’:PTX:CDN was 5:1:0.1 at all concentrations. n=2–3 replicates of a representative batch. (l) Fluorescence intensity of Cy7-CDN indicating tumor retention of CDN. Free CDN* or 2E’/PTX/CDN* (5:1:0.1, w/w/w) was intratumorally injected into CT26 subcutaneous tumor at a total dose of 0.02 mg CDN* (CDN + Cy7-labeled CDN in 1:1 (w/w) ratio). The intensity of Cy7-CDN fluorescence signal was monitored by the Spectral Ami Optical Imaging System over 7 d from the injection and expressed as values normalized to the intensity at the time 0, n=2 for free CDN; n=3 for 2E’/PTX/CDN, mean ± SD. An extended data set with the full timescale and replicates is shown in Fig. S7 . P values in d-f were calculated by Tukey’s multiple comparisons test following ordinary one-way ANOVA.

Article Snippet: The fluorescence signal of CDN was monitored by the Spectral Ami Optical Imaging System (Spectral Instruments, Tucson, AZ) over 7 d from the injection.

Techniques: In Vitro, Derivative Assay, Incubation, Concentration Assay, Labeling, Membrane, Staining, Fluorescence, Flow Cytometry, Activation Assay, Positive Control, Negative Control, Injection, Optical Imaging

Journal: bioRxiv

Article Title: An autologous cell-based therapeutic vaccine expressing IL6/1 fusokine drives robust anti-tumor response against ovarian cancer

doi: 10.64898/2026.05.05.721149

Figure Lengend Snippet: (A) Schematic representation of the experimental design and treatment schedule for in vivo study of mIL6/1 engineered cell therapy in ovarian cancer mouse model. (B) Tumor growth was quantitatively analyzed using bioluminescence (BLI) signals obtained from LAGO imaging system at a week after tumor inoculation and subsequent weeks. Longitudinal images of a representative mouse from each treatment group were presented from a week after tumor inoculation before treatment and subsequent weeks. (C) Total emission signal (photons/sec) were calculated by Aura software and normalized to the signal before start of treatment. Statistical significances were analyzed using Two-Way ANOVA followed by Tukey’s multiple comparison tests. Data were indicated as the mean ± SEM or Student T tests. (D) Spaghetti plot for each mouse in all experimental groups are shown. (E) Kaplan-Meier survival curves for mIL6/1 cell therapy. Mice were considered dead if found dead in their cage or predefined endpoints due to health conditions or abdominal distension. (F) Tumor growth as shown by luciferase signal measured using Bioluminescence (BLI) method and the signal measured (photons/sec) were calculated by Aura software and normalized to the signal before start of treatment. Statistical significances were analyzed using Two-Way ANOVA followed by Tukey’s multiple comparison tests. Data are presented as the mean ± SEM. (G) Kaplan-Meier survival curves for ID8IL6/1 vs ID8EV cell therapy. Mice were considered dead if found dead in their cage or predefined endpoints due to health conditions or abdominal distension.

Article Snippet: ID8-F3 cells stably expressing luciferase (ID8 Luc + ) were used to monitor tumor progression and quantify tumor burden via in vivo bioluminescence imaging system, the Lago Spectral imaging system (Spectral Instruments Imaging).

Techniques: In Vivo, Imaging, Software, Comparison, Luciferase