Fig. 4: Micellar peritumoral cAMP suppression prevents tumor immune cell dysfunctionality.

A Tumor volumes, experimental workflow, and unsupervised cell clustering. C57BL/6 mice were subcutaneously inoculated with B16-OVA tumor cells and received peritumoral micelle injections every other day starting from day 4 after inoculation. Tumor volumes shown as mean ± SEM, n = 10 per group, NL-PM (blue) vs. L-PM (red): **P = 0.0079 (day 11), **P = 0.0084 (day 13), *P = 0.0196 (day15); (parametric unpaired two-sided t-test with Holm–Šídák-corrected P values, alpha = 0.05, 0.95 confidence interval). At day 15, CD45⁺ single cells were isolated from tumor tissue and subjected to single-cell transcriptome profiling. 2D tSNE plots of color-coded cell populations and pie charts with numbers of clustered cells (monocytes/macrophages, orange; neutrophils, red; dendritic cells (DC), light blue; plasmacytoid DC, green; natural killer (NK) cells, dark blue; T cells, yellow; B cells, gray). B AUC scores using an anti-inflammatory input gene set (Ptgs2, Vegfa, Egfr, Arg1, Ccl22, Ccl17, Il10, and Il12a) on monocytes/macrophages and neutrophils treated with NL-PM or L-PM. *P = 0.024, ***P = 0.001 (non-parametric unpaired two-sided Mann–Whitney test). C Heatmap showing z-scored average expression of indicated exhaustion marker genes in T cells treated with NL-PM or L-PM (left) and AUC score using the heatmap’s exhaustion marker gene set as input on NL-PM or L-PM treated T cells (right). ***P = 0.00099 (non-parametric unpaired two-sided Mann–Whitney test, alpha = 0.05, 0.95 confidence interval). Data are shown as box and whiskers plots. Centerlines represent the median, the edges of the box indicate the 25th and 75th percentiles and the whiskers represent the 10th and 90th percentiles. Single-cell RNA-Seq data are accessible via NCBI Gene Expression Omnibus (GEO) database accession number: GSE166028 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166028].