Fig. 4

TTS treatments increased the number of M1 macrophages and CD8 T-cell abundance and activity. A Graph displaying CD8/TAM ratios in the TME of MC38-hEpCAM tumors after the completion of each treatment cycle, according to FC analysis (left) and NanoString gene expression analysis (right). The TAM (CD11B⁺CD11C⁻F4/80⁺) FC gating strategy can be found in Additional file 1: Fig. S1B. B Representative pseudocolor plots showing MHC II and CD206 expression in CD11b⁺ F4/80⁺ TAMs on treatment cycle 3 as determined by FC for the detection of TAM M1 and M2 subsets as described in the upper left legend. C Violin plots show the tumor density (cells/mg tumor) of CD206^lowMHCII^highM1-TAMs (left) and CD206^highMHCII^lowM2-TAMs (right) over the treatment cycles as determined by FC. D Violin plots show the tumor M1 to M2 TAM ratios over the study period. A–D n = 3–4/group/cycle; timepoints are referred to as C1-C4 (cycles 1–4, respectively). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control, for C4 Combo versus C215Fab-SEA. E Heatmap displaying the mean log2-fold expression values of changed genes (left) associated with M1 macrophage activity in tumors upon different treatments over the treatment cycles. n = 3–4/group/cycle. The color gradient indicates the fold increase relative to the matched control. White represents a nonsignificant expression change compared to the matched control. Differential expression analysis was performed with Welch’s t test