Fig 2: Distinct mesenchymal phenotypes and transcriptional state-specific immune heterogeneity exist in the liver metastatic microenvironment(A) t-SNE visualization of non-malignant cells identified in the metastatic microenvironment, abbreviations as in Figure S1J (TAM, tumor associated macrophage).(B) Expression of Fibroblast-like (PC2 low) and Pericyte-like (PC2 high) mesenchymal (Mes.) cell programs across different metastatic sites (top).(C) Density plots for mesenchymal cell phenotype score in single cells from our metastatic cohort (top) or previously published PDAC bulk RNA-seq profiles (bottom), fill indicates anatomic site. P-value determined by Student’s t test (top) or by ANOVA followed by Tukey’s HSD (bottom).(D) Summary of mesenchymal phenotypes in primary versus liver metastatic PDAC.(E) Correlation between microenvironment diversity (Simpson’s index, x axis) and the average malignant scBasal-scClassical commitment score (y axis) for each scRNA-seq sample.(F) Correlation between TME diversity as inferred by immunogenic signature score (x axis) versus average tumor scBasal-scClassical commitment score (y axis) in primary and metastatic bulk RNA-seq samples.(G and H) Sample-level (columns) variation in Simpson’s index (G, dot plot), average malignant scBasal-scClassical (G, top heat bar) and IC (G, bottom heat bar) expression, and fraction of non-malignant cell types (H). Samples were clustered and ordered within metastatic site (liver versus other) by their fractional representation of cell types. Dots indicate top statistically significant cell type frequency differences calculated using a Kruskal-Wallis test with multiple hypothesis correction.(I) Boxplots compare cell type fractions between the scBasal predominant tumors with low diversity (PANFR0593, 575, 545) and all others. P-value determined by Student’s t test.(J) Summary of associations between microenvironmental diversity, non-malignant cell types, and malignant cell state.See also Figure S6; Table S2.

Fig 3: IHC analysis of original and xenografted tumors. The expression of ERa and PR was decreased in 4Z-B-1 cell xenografts, while that of EGFR and Ki-67 was elevated. The expression of HER2, P120, CK5/6, and E-cadherin was similar in preoperative puncture specimens, operation specimens and 4Z-B-1 xenografts. Magnification: ×200.

Fig 4: Organ-like structures of the NBOs and LCOs. a Bright-field microscopy images of a NBO, NBO-90. NBO-90 was cultured in MBM and MBM + WNA (W; Wnt3A, N; Noggin, A; A83-01). Scale bars, 200 µm. b H&E staining image of NBO-90. Scale bar, 100 µm. c–e Immunofluorescence images showing the expression of pancytokeratin, p63, MUC1, KRT5, KRT7, CC10, and cilia co-stained with acetylated a-tubulin (Ac-Tub) and Arl13b (The enlarged images in yellow boxes showed cilia co-stained with Ac-Tub and Arl13b). Nuclei (blue) were stained with DAPI. Scale bar, 20 µm

Fig 5: Ras/ERK and PI3K/AKT pathway regulation of ERG result in distinct gene expression programs in prostate cells.(A) Heat map comparison of genes in RWPE1-ERG S96E or RWPE1-ERG/mAKT significantly differentially regulated compared to RWPE1-ERG alone. Gene ontology analysis of co-regulated genes (green) and differentially regulated genes (red). (B) Immunoblot of RWPE1 xenograft tumor lysates comparing those expressing only ERG-S96E and those expressing ERG and mAKT. (C) Immunoblot of RWPE1 xenograft tumor lysates expressing phospho-mutants of ERG. (D) Histology of RWPE1-ERG S96E or RWPE1-ERG/mAKT xenograft tumors. Top images stained for KRT5 (basal) and KRT8 (luminal) markers and bottom images stained for Pan-CK and VIM.