Multi-omics profiling unveils biological and clinical insights into pulmonary sarcomatoid carcinoma

Figure 1. Multi-omics landscape of PSC (A) Workflow of PSC multi-omics profiling. (B) Genetic profiles in the TU-PSC cohort. The significantly mutated genes with q value <0.1 are indicated in red. (C) Mutation frequency of somatic mutations across four PSC cohorts. (D) Kaplan-Meier curves comparing overall survival for KRAS-mutant and KRAS wild-type (WT) patients (log rank test). (E) Kaplan-Meier curves comparing overall survival for high TMB and low TMB patients (log rank test). (F) Trinucleotide motif frequency plots and enriched mutational signatures identified in the TU-PSC cohort.

Figure 2. Effects of genetic alterations on proteogenomic profiles in PSC (A) The Venn diagram showing the significant cis-events restricted to cancer-associated genes (CAGs) across multiple data types. (B) Enriched pathway for 16 cis CAG-related significant cis-events shared at mRNA, protein, and phosphoprotein levels. (C) Genome-wide arm amplification and deletions. (D) Genome-wide focal amplification and deletions. Chromosomal locations of peaks of significantly recurring focal amplifications (red) and deletions (blue) were filtered by FDRs. (E) Boxplot showing the KDM5D mRNA expression differences between Yq amplification and Yq non-amplification tumors (Wilcoxon ranked-sum test). (F) Boxplot showing the PTEN protein differences between PTEN-mutant and PTEN wild-type (WT) tumors (Wilcoxon ranked-sum test). (G) Boxplots showing the SMARCA4 expression differences between SMARCA4-mutant and SMARCA4 WT tumors (Wilcoxon ranked-sum test). (H) Heatmap of SMARCA4 phosphosites between SMARCA4-mutant and SMARCA4 WT tumors. (I) Significantly upregulated pathways in SMARCA4-mutant tumors compared with SMARCA4 WT tumors via gene set enrichment analysis. (J) Boxplots showing the PRC2 complex protein…

Figure 3. Proteomic and phosphoproteomic alterations associated with tumorigenesis and prognosis (A) The differential protein expression measured using FDR and log2 fold-change (FC) between tumors and NATs (Wilcoxon rank-sum test, proteins quantified in all samples). (B) Representative enriched biological processes of increased (left) and decreased (right) differentially expressed proteins in tumors compared with NATs. (C) Boxplot showing protein expression differences measured in log2 FC between tumors and paired NATs for representative 50 PSC-associated proteins according to the median log2 FC in paired samples, the potential clinical utilities of which were annotated based on the Human Protein Atlas. CRG, cancer-related gene. (D) Boxplots showing the SRSF5 mRNA (left) and protein (right) expression differences between tumors and NATs (Wilcoxon rank-sum test). (E) The differential expression at phosphorylation site level measured using FDR and log2 FC between tumors and NATs (Wilcoxon rank-sum test, phosphorylation sites quantified in half samples). (F) Comparison of abundance changes (log2 FC) in tumors between protein level and phosphorylation site level (quantified in half samples).…

Figure 4. Derepression of ferroptosis decreases cell proliferation in PSC (A) Western blot showing the knockdown efficiency of SLC3A2 and FTL in PLA-801D cells. (B) The cell proliferation of PLA-801D cells measured using CCK-8 assays following the knockdown of SLC3A2 (left) and FTL (right), respectively. (C) The cell proliferation of PLA-801D cells measured using colony formation assays following the knockdown of SLC3A2 (upper) and FTL (bottom), respectively. Following the knockdown of SLC3A2 or FTL, PLA-801D cells were plated and cultured for 14 days. All colonies were subsequently stained with crystal violet and quantified using ImageJ software. (D) The extracellular GSH concentration of PLA-801D cells was tested following the knockdown of SLC3A2. (E) Following the knockdown of FTL, PLA-801D cells were stained by FerroOrange and quantified using the microplate reader, respectively. (F) The lysates of PLA-801D cells were immunoblotted with the indicated antibodies following the knockdown of FTL. (G) Following the knockdown of SLC3A2, PLA-801D cells were treated with 10 μM ferrostatin-1 for 24 h. Afterward, the extracellular GSH concentration was tested. (H) Following the knockdown of FTL,…

Figure 5. The immune landscape of PSC (A) Heatmaps showing compositions of cell types, mRNA expression of representative immune-related markers, and activities of enriched pathways across three immune clusters: hot tumor cluster, cold tumor cluster, and NAT-enriched cluster (successive heatmaps illustrate xCell immune signatures, mRNA expression of key immune-related markers, and single-sample GSEA (ssGSEA) pathway scores based on mRNA expression for biological pathways that were differentially regulated in immune cluster). (B and C) Comparison of immune score (B) and TIDE score (C) between hot tumor cluster and cold tumor cluster (Wilcoxon rank-sum test). (D) Significantly activated pathways at protein level in hot tumors compared with cold tumors via gene set enrichment analysis. (E) Comparison of PSC histological subtype distribution between hot tumor cluster and cold tumor cluster (Fisher’s exact test). (F) Comparison of PTEN mutation rate between hot tumor cluster and cold tumor cluster (Fisher’s exact test). (G) Comparison of immune score between PTEN-mutant and PTEN wild-type (WT) tumors (Wilcoxon rank-sum test). (H) Comparison of xCell score of immune cells between PTEN-mutant and…

Figure 6. Proteomic subtypes and molecular characteristics of the TU-PSC cohort (A) Heatmap showing clinicopathologic features, representative enriched biological processes, and signature proteins of three proteomic subtypes identified using NMF-based unsupervised clustering. (B) Heatmap showing significantly enriched pathways (MSigDB Hallmark) in three proteomic subtypes via gene set enrichment analysis. ∗p < 0.05, ∗∗p < 0.01. (C) Stacked bar plot showing the distribution of PSC immunotype (hot tumor and cold tumor) in each proteomic subtype (chi-squared test). (D) Comparison of PD-L1 mRNA (left) and protein (right) expression among three proteomic subtypes (one-way ANOVA following Tukey’s test). (E) Significant enrichment of proteasome and proteasome complex pathways in the S1 subtype via gene set enrichment analysis. (F) Comparison of protein expression of PSMB8 among three proteomic subtypes (Wilcoxon rank-sum test). (G) Comparison of protein expression of PSMB9 among three proteomic subtypes (one-way ANOVA following Tukey’s test). (H) Significant enrichment of antigen processing and presentation pathway in protein level in the S1 subtype via gene set enrichment analysis. (I)…

Figure 7. scRNA-seq profiles for PSC with ICB immunotherapy (A) UMAP plot of all scRNA-seq data showing 12 cell types. (B) Heatmap showing the expression of top marker genes of 12 cell types. (C) Expression comparison of EMT-related genes (VIM, COL1A1, COL6A1, and TIMP1) between carcinoma and sarcomatoid cells (Wilcoxon rank-sum test). (D) Expression comparison of EMT-related transcription factors (TFs) (ZEB2 and SNAI2) between carcinoma and sarcomatoid cells (Wilcoxon rank-sum test). (E) Expression comparison of epithelial genes (KRT7 and EPCAM) between carcinoma and sarcomatoid cells (Wilcoxon rank-sum test). (F) Expression comparison of MHC-II genes (HLA-DMA, HLA-DRA, HLA-DRB1, and HLA-DPB1) between carcinoma and sarcomatoid cells (Wilcoxon rank-sum test). (G) Significantly activated pathways in NMPR tumors compared with pCR tumors via gene set enrichment analysis. (H) Expression comparison of EMT-related genes (VIM, COL1A1, COL4A1, and COL6A1) between NMPR and pCR tumors in baseline samples (Wilcoxon rank-sum test). (I) Comparison of C1 program score between NMPR and pCR tumors in baseline samples (Wilcoxon rank-sum test). (J) The significant enriched KEGG pathways for the C1 program.…