Fig 2: Comparative analysis between the standard method and PeptiCHIP technology. (A) Peptide length distribution is reported for the standard method (blue bars) and the microchip (green bars) for each number of cells (left, 1 × 106 cells; right, 10 × 106 cells). (B–E) The 9mers consensus binding motifs were deconvoluted by unsupervised Gibbs clustering analysis for both the standard method and the microchip and for each number of cells. The reference motif is shown in the upper right corner. The clusters with higher KLD values were chosen (orange star), and the sequence logo is reported. (F–I) The binding affinities to HLA-A*02:01 and HLA-B*07:02 were predicted for the eluted 9mers, and the percentages of binders and not binders are depicted as a part to the whole for both the standard method and the microchip and for both numbers of cells.

Fig 3: In depth enrichment analysis of HLA-ligands source proteins and CD8+ T-cell based cytotoxic assay. (A) Gene ontology enrichment analysis of the HLA-ligands source proteins. The most overrepresented biological processes for 10 × 106 cells are separately shown for HLA-A*02:01 and HLA-B*07:02 alleles (hypergeometric test padj <0.01). (B) Molecular Signature Database results are displayed. The source proteins analysis was performed against the hallmark data set and separately for HLA-A*02:01 and HLA-B*07:02 alleles.

Fig 4: “Bulk” RNA sequencing of 2D and 3D cultured GBM models. (A) Principal component analysis of 11 GBM models. “Distinct” 3D (Group 1, Group 2) and corresponding 2D (Group 3, Group 4) samples are highlighted. Percent of differentially regulated genes of the groups are indicated. “Non-Distinct” (N.D.) 2D and 3D models are indicated. (B) Volcano plot of differentially regulated genes (2D versus 3D) with a log fold change >5 and p-value < 0.05. (C) Heat map of the top 50 most variable genes. Corresponding cultures are labeled 3D (solid box) and 2D (dotted line). (D) GSEA with enrichment plots for “Interferon α/β” (R-HSA-909733) signaling and “Antigen presentation, folding assembly and peptide loading of class I MHC” (R-HSA-983170) pathways using the Reactome Pathway Database (FDR score < 25%). (E) Normalized expression values for selected MHC class surface receptor genes (HLA-A, HLA-E), B2M, and stress-signaling markers (MICA and 14-3-3ε/14-3-3ζ). Asterisks indicate significant differences with p-value < 0.05.