Fig. 5. In vivo antitumour activity of PEG-T^ECM-NS/OLE in Balb/c mice bearing 4T1 breast tumour.

a In vivo fluorescence imaging after intravenous injection of Cy5.5-labelled PEG-T^ECM-NS without crosslinked structure (as control group), PEG-T^ECM-NS, DSPE-PEG, and PEG-PCL. b Quantitative analysis of fluorescence signal (n = 5 biologically independent mice), scale bar 2 cm. c Blood clearance curves of OLE after intravenous injection of different formulas (n = 5 biologically independent mice). d Biodistribution of OLE in tumour after intravenous administration of different treatments. Estimation of OLE in tissues was performed using high performance liquid chromatography (HPLC) (n = 5 biologically independent mice). e T₁-weighted MRI of the 4T1 tumour-bearing mice after tail vein injection of PEG-T^ECM-NS without crosslinked structure as control group and PEG-T^ECM-NS modified with Gd at a dose of 10 mg kg⁻¹ body weight, scale bar 1 cm. f PA imaging of the 4T1 tumour mice after different treatments, scale bar 1 cm. The individual (g) and averaged (h) 4T1 tumour growth curves after different treatments (n = 5 biologically independent mice). i Body weight of 4T1 tumour mice treated with different formulations (n = 5 biologically independent mice). j Cumulative survival curves of tumour mice. k Immunofluorescence of HMGB1, CRT, and CD8⁺ T cells in the tumour tissues after different treatments (left) and quantitative analyses (right) (n = 25 independent replicates). l Quantification of secretion of IL-12p40, IFN-γ, and TNF-α in sera from mice isolated from groups of mice on day 7 (n = 10 biologically independent mice). m Western blot analysis of HMGB1 and CRT expression levels in the tumour tissues. The samples were derived from the same experiment and the gels were run in parallel. n Ki67 staining and H&E staining of the tumour tissues with different treatments. o The determination of IFN-γ positive CD8⁺ T cells (CD8⁺IFN-γ⁺ T cells) within tumour by flow cytometry (left) and quantitative analysis (right) (n = 5 biologically independent mice). p The percentage of Tregs (CD4⁺Foxp3⁺ T cells) within tumour by flow cytometry (left) and quantitative analysis (right) (n = 5 biologically independent mice). For the boxplots (o, p), the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, and whiskers represent ±1.5 inter-quartile range. q CD8⁺ T cells in 4T1 tumour analyzed by flow cytometry. r T-distributed stochastic neighbour embedding (t-SNE) visualization of clustering of representative markers of cells from tumour detected by flow cytometry, each dot corresponds to one single cell. All error bars represent mean ± s.d. Statistical significance was evaluated using Student’s unpaired t test. Asterisks indicate p values *P < 0.05, **P < 0.01, and ***P < 0.001 and n.s. represents no significant difference.