Fig. 4

Tumor infiltration with immunosuppressive cells of the monocytic lineage after SBRT associates with progression. a and b CD68/CD163 co-labeling assessed by multiplexed immunohistochemistry on tumor FFPE biopsies. CD68+/CD163− (a) and immunosuppressive CD68+/CD163+ (b) cell densities were automatically quantified by the HALO® software on digitalized slides. Are displayed: cell density at baseline according to treatment response (left panel), cell density according to biopsy timepoint among patients with progressive disease (middle panel), and among patients with stable or responsive disease (right panel). Blue and yellow crosses refer, respectively, to patients with Progressive and Stable+Elite disease. Mean ± 95%CI. Kruskal–Wallis with Dunn’s multiple comparisons test. c Differential gene expression analysis of RNA-seq data comparing transcription profiles of tumor biopsies from patients who progressed with patients who showed stable disease or tumor response among 1775 selected genes. Genes differentially expressed between groups with adjusted P-value < 0.05 are indicated by yellow or blue crosses, respectively when downregulated or upregulated in progressive patients. Relevant genes implicated in immune regulation are depicted. d–f Heatmap depicting scores per immune cell type (y-axis) according to subgroups classified according to treatment response and biopsy timepoint (x-axis) using two independent published deconvolution tools, CIBERSORTabs (f) and xCELL (e). Scores are normalized per cell type based on the calculation: mean of the subgroup/total mean. Blue indicates higher values than the mean of samples, while pink indicates lower values. Stars indicate significant P-values obtained from the comparison of ’Progressive’ versus ‘Stable+Elite’ among samples obtained at the indicated timepoint, Kolmogorov–Smirnov test. f Idem, deconvolution of bulk RNA-seq data using the MoMac-VERSE obtained from single-cell RNA-seq description of 17 mononuclear cell types¹⁶. Scores are normalized per cell type based on the calculation: mean of the subgroup/total mean. Blue indicates higher values than the mean of samples, while pink indicates lower values. Stars indicate significant P-values obtained from the comparison of ’Progressive’ versus ‘Stable+Elite’ among samples obtained at the indicated timepoint, Kolmogorov–Smirnov test. Crosses indicate missing values. *P-value ≤ 0.05; **P-value ≤ 0.01; ***P-value ≤ 0.001