Fig 2: Spatial profiling across samples. (A) Confocal microscopy images: overlaying nuclear staining (blue), NY-ESO-1 (yellow), pan-cytokeratin (green), and CD45 (red) were used to identify ROIs, indicated by circles. Pink circles in the metastasis lesion indicate those that were within the tumor region. (B) Heatmap of the Spearman rank correlation across all matched protein–RNA markers for all ROIs assessed. Each ROI is indicated by the corresponding biopsy and assay by the color bars at the bottom of the plot. (C) Heatmap of the marker-scaled expression of proteins (top) and genes (bottom) assessed across ROIs; non-tumor regions from the metastasis are not included in this figure. Markers were clustered by assay type and scaled expression using unsupervised hierarchical clustering with Ward agglomeration. Markers are also annotated on the right by corresponding hallmark gene sets. (D) Raw expressions of CTNNB1, CD8/CD8A, and CD4/CD4 are shown on the y-axis across ROIs derived from each sample (denoted by color). (E) The Spearman rank correlation (y-axis) was quantified across all matched protein or RNA markers across ROIs and between ROIs and matched bulk RNA samples. Comparisons are indicated along the x-axis. (D, E) Pairwise Wilcox tests were performed across groups. Non-significant comparisons are not shown; significant differences are indicated across comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DSP, digital spatial profiling; ROI, region of interest;

Fig 3: Representative micrographs of cores on a tissue microarray stained for (A) PRAME, (B) NY-ESO-1, and (C) SSX2. Numbers represent semiquantitative scoring of immunostaining: 0, negative; 1, weak; 2, moderate and 3, strongly positive. Magnification 20×.

Fig 4: Activation of innate immunity by NY-ESO-1-expressing artificial adjuvant cells (aAVC-NY-ESO-1). A, aAVC-NY-ESO-1 cells were established by loading NIH3T3 vector cells with a-galactosylceramide (a-GalCer) and coelectroporation with NY-ESO-1 and murine CD1d mRNA. B-D, NY-ESO-1 protein levels and CD1d surface expression were assessed using western blotting (B) and flow cytometry (C), respectively (red, aAVC- NY-ESO-1; bold, NIH3T3; shaded, isotype). D, a-GalCer presentation by aAVC-NY-ESO-1. aAVC-NY-ESO-1 were cocultured with the Va14 invariant natural killer T (iNKT) cell hybridoma 1.2 for 24 h, and interleukin-2 (IL-2) production in the culture supernatant was evaluated by ELISA (n = 4; mean ± SEM) ***P < .001 (Mann-Whitney). E, iNKT and natural killer (NK) cell activation. Mice were injected with 5 × 105 aAVC-NY-ESO-1 i.v. Spleens were removed 6 h later, and iNKT as well as NK cells were analyzed by staining with CD19-antigen presenting cells (APC) and CD1d-dimer+ phycoerythrin (PE) or CD3-FITC and NK1.1-APC. These cells were simultaneously stained with either surface CD69-PE or intracellular interferon-?-PE and evaluated through flow cytometry (n = 4). F, Antitumor immunity by innate lymphocytes. Mice were injected with 3 × 105 B16 melanoma cells i.v. and, 3 h later, were inoculated with 5 × 105 aAVC-NY-ESO-1. The number of lung metastases was determined 14 d later (n = 5 per group, mean ± SEM). In some experiments, NK cells were depleted using anti-asialo GM1 Ab. ***P < .001 (Tukey’s test) (aAVC-immunized vs others)

Fig 5: Univariate analysis of overall survival depicted as Kaplan–Meier curves stratified according to the expression of (A) PRAME, (B) NY-ESO-1, and (C) SSX2.