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. 2019 Feb 28;116(12):5223–5232. doi: 10.1073/pnas.1819303116

Fig. 1.

Fig. 1.

Single-cell transcriptome profiling of STCs and lung metastases. (A) Schematic representation of the orthotopic mouse cancer model, with representative sections of the primary tumor and STCs in the lungs (STC6 and STC9–11) after prostate inoculation. The primary GFP-tagged CE1-4 mouse prostate cancer cells are derived from tissue-specific inactivation of Pten in a mouse model (23). Tumor cells are identified by IHC staining for GFP, and proliferative cells are scored by dual-IF staining for GFP and Ki67. (Insets: Magnification 3×.) (Scale bars, 25 μm.) (B) Unsupervised hierarchical clustering of single-cell RNA-Seq of 149 mouse prostate tumor cells. GFP-tagged primary tumor cells (n = 29), CTCs isolated by microfluidic capture (24) from blood specimens (n = 12), STCs and fewer than six cell clusters collected from the lungs at STC6 (n = 20) and STC9–11 (n = 55), and micrometastases evident at 9–11 wk (Met1 and Met2, n = 33) were individually micromanipulated and subjected to single-cell RNA-Seq. The genes displayed are the top 2,000 genes with respect to variance across the samples of the RPM values. (C, Left) Scatter plot showing the cell cycle metascore (G1/S + G2/M) of tumor cells collected from the lungs at STC6 and STC9–11 after orthotopic inoculation (***P < 0.001, two-tailed Student t test). (C, Right) Bar graph showing gene set enrichment for cell cycle pathways in single-cell RNA-Seq of tumor cells collected from the lungs at STC9–11 compared with those collected at STC6 (x axis: −log10 of P value). (D, Left) Scatter plot showing the cytokine signaling metascore of STCs collected from the lungs (STC6 and STC9–11) after orthotopic inoculation (***P < 0.001, two-tailed Student t test). (D, Right) Bar graph showing gene set enrichment for cytokine pathway clusters based on single-cell RNA-Seq of STC9–11 compared with STC6 (x axis: −log10 of P value). (E) Heat maps of the cell cycle pathway genes (Top) and cytokine signaling pathway genes (Bottom) up-regulated within STC9–11 single cancer cells compared with STC6 single cancer cells.