Fig. 2.

Prolactin receptor signaling induces metastatic outgrowth. (A) Scatter plot showing the median single-cell RNA-Seq expression for cytokine and growth factor receptors (statistical threshold: P < 0.05) versus log-twofold change between STCs collected from the primary tumor and lungs after 6-wk orthotopic inoculation (STC₆) versus 9- to 11-wk orthotopic inoculation (STC_9–11) and micrometastases. Prlr is the most abundant differentially expressed receptor. (B) Scatter plot showing single-cell RNA-Seq expression of Prlr in dissociated primary tumor cells, STCs in the lungs after 6 wk (STC₆) and 9–11 wk (STC_9–11) of tumorigenesis, and dissociated micrometastases. The dashed line represents the threshold of 500 RPM (**P < 0.01, nonparametric Mann–Whitney U test). (C) Representative IF images showing the expression of Prlr in lung metastases from three prostate cancer orthotopic mouse models expressing endogenous Prlr, compared with normal lung (Ctrl). (Scale bars, 25 μm.) (D) Western blot analyses showing Dox-inducible expression of Prlr in engineered CE1-4 murine prostate tumor cells and activation of its downstream STAT-5 signaling following treatment with 100 ng/mL mouse Prl for 5 or 15 min. (E) In vitro growth curves of CE1-4 cells cultured in the presence or absence of Dox to induce Prlr expression, with or without addition of Prl. A modest antiproliferative effect is evident in cells expressing Prlr and treated with Prl (**P < 0.01, two-tailed Student t test). V, vehicle. (F) Schematic representation of Dox-inducible expression of Prlr within prostate tumors derived from orthotopic injection of CE1-4 cells. In the presence of Dox, Prlr is detectable by RNA-ISH within the primary tumor cells, as well as within small micrometastases in the lungs. (Scale bars, 50 μm.) (G) Bar graph shows reduced size of the primary tumor following the induction of Prlr expression with Dox (+), compared with V-treated controls (−) (*P < 0.05, two-tailed Student t test). (H) Bar graph shows quantitation of lung tumor foci across different size categories (one to five cells, six to 20 cells, >20 cells) in Dox-induced (+) versus V-treated (−) mice 6 wk after tumor inoculation (n = 3 mice per group with at least 10 fields quantified per animal). Post hoc power of 0.16 (***P < 0.001, **P < 0.01; two-tailed Student t test). n.s., not significant.