Fig 2: SLFN11 protein levels in HGSOC and their correlation with TILs.(A) Correlogram of TILs and SLFN11 H-scores, assessed in overall, cancer, and noncancer cells. In the lower triangle of the graph, boxes represent pairwise correlations colored by direction (blue for negative correlations and red for positive ones) and strength (intensity of shading) of the correlation itself. In the upper triangle, circles use the same scaled colors, but fill an area proportional to the absolute value of the correlation, and are filled clockwise for positive values, counterclockwise for negative values. (B and C) Scatterplots representing total CD3+ cells (B) and total CD8+ cells (C) — (y axes, cells/mm) as a function of SLFN11 H-score in noncancer cells (x axis); ? is the Spearman’s correlation coefficient; the least squares regression are represented by the red lines, whereas dots are measurements of immune cell counts by H-scores in individual samples. (D) Representative images of SLFN11 IHC in HGSOC specimens. Left, stroma SLFN11hi and tumor SLFN11lo; right, stroma and tumor SLFN11hi, for the indicated cancers. The images highlight nuclear SLFN11 protein localization in tumor cells and different stromal cell subtypes. Scale bars: 50 µm.

Fig 3: SLFN11 is expressed in a subset of immune-related cells and is associated with immune signatures in HGSOC.(A) Multiplex immunofluorescence for SLFN11 in T cells (CD3+CD8+), B cells (CD20+), and macrophages/monocytes (CD68+) infiltrating HGSOC tissue. Scale bars: 50 µm. (B) Dendrogram representing the similarity between different immunologic signatures calculated in TCGA ovarian cancer data set (n = 302 cases). X axis represents the Ward’s D2 distance. Signature names have superscript numbers to denote the publication they are derived from: 1: (32); 2: (31); 3: (30); 4: (38); 5: (28); if not specified, signatures are from 6: (27). Right: lollipop plot of correlations between SLFN11 and the aforementioned signatures. X axis represents the Spearman’s correlation coefficient between SLFN11 expression in TCGA ovarian cancer data set and the investigated signatures, whereas individual dots are colored by –log10 of the FDR of the correlation.

Fig 4: SLFN11, immune cell subpopulations, and prognosis in TCGA serous ovarian carcinoma data set.(A) Variable correlation plot of the PCA including SLFN11 transcript, cancer cellularity, and CIBERSORTx immune cell subpopulations significant by univariable correlation at FDR < 0.05. The 2 axes represent the first principal components explaining the greatest fraction of the variance of the analyzed data set, with percentage of explained variability in parentheses. The relative position of the variables toward each other explains their relative correlation, whereas their distance from the intersect accounts for their contribution to the components. SLFN11 is represented with a thick red arrow for sake of clarity. (B) Kaplan-Meier plot showing PFI stratified by SLFN11 transcript (“high” if above the lower tertile of expression in the data set, “low” if below). The progressed fraction of patients (y axis) is plotted against time expressed in months from the end of first-line CT, censored at 60 months (x axis). Numbers at risk are reported below the plot. P value in the bottom left of the plot is from the Wald statistics for the univariable Cox’s regression. (C) Forest plot of hazard ratios (x axis, in log scale) for variables retained in the lasso-selected multiple Cox’s regression model. Point HR estimates are reported below each variable together with 95% CIs in parentheses, whereas adjusted P values for each variable are on the right side of the plot. Filled black squares represent HR estimates, with relative 95% CIs shown as horizontal lines with brackets.

Fig 5: SLFN11 is a dual biomarker capturing simultaneously interconnected immunological and cancer cell–intrinsic functional dispositions associated with sensitivity to platinum treatment.(A) Bubble chart representing significantly enriched pathways during treatment with cisplatin in SLFN11hi (n = 3) versus SLFN11lo (n = 4) ovarian cancer cell lines. The bubble size indicates the q value (FDR), whereas the color represents the direction of the change as normalized enrichment score (NES), with red indicating a positive enrichment of the comparison and blue a negative one. c, concentration. (B) Heatmap of log fold changes of selected immune-related transcripts for the comparison between the SLFN11hi and SLFN11lo cancer cell lines at the same time points as for A. (C) PBMCs were incubated with IFN-ß, GM-CSF, or anti-CD3/CD28 beads for 24 hours and immunoblotted as indicated. Chart shows SLFN11 densitometry (n = 4). (D) DU145 parental or SLFN11-KO cells were stained with/without anti-SLFN11 and analyzed by flow cytometry. (E) PBMCs were treated as in C and analyzed by flow cytometry. FACS plots show SLFN11 and CD25 expression in naive (top) and anti-CD3/CD28-stimulated (bottom) cultures. Charts show quantification of SLFN11hi CD4+ and CD8+ T cells (top) and CD25 expression in SLFN11hi/SLFN11lo T cells (bottom). (F) Purified CD3+ T cells were stimulated with anti-CD3/CD28 beads and immunoblotted as indicated. Chart shows SLFN11 densitometry (n = 4). (G) PBMCs were treated as in C and analyzed by flow cytometry for SLFN11 and programed cell death ligand 1 (PD-L1) expression in myeloid cells. Chart shows quantification of SLFN11hi myeloid cells (n = 8). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. All data in graphs are presented as mean ± SEM. Statistics by Student’s t test, pairwise comparisons with the 2-tailed t test with reported P values adjusted for family-wise error using the Holm’s method. *P < 0.05; **P < 0.01; ****P < 0.0001.