Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation

Abstract

We have developed a screening platform for the isolation of genetic entities involved in metastatic reactivation. Retroviral libraries of cDNAs from fully metastatic breast-cancer cells or pooled microRNAs were transduced into breast-cancer cells that become dormant upon infiltrating the lung. Upon inoculation in the tail vein of mice, the cells that had acquired the ability to undergo reactivation generated metastatic lesions. Integrated retroviral vectors were recovered from these lesions, sequenced, and subjected to a second round of validation. By using this strategy, we isolated canonical genes and microRNAs that mediate metastatic reactivation in the lung. To identify genes that oppose reactivation, we screened an expression library encoding shRNAs, and we identified target genes that encode potential enforcers of dormancy. Our screening strategy enables the identification and rapid biological validation of single genetic entities that are necessary to maintain dormancy or to induce reactivation. This technology should facilitate the elucidation of the molecular underpinnings of these processes.

Keywords: cDNA library screen; forward genetic screens; metastatic reactivation; microRNA library screen; shRNA library screen.

Fig. 1.

Screening platform for the isolation of genes that mediate metastatic reactivation. (A) Schematic representation of the metastatic capabilities of the mouse mammary carcinoma cells constituting the progression series used here. (B) 4TO7-TGL cells transduced with empty vector or Coco were inoculated i.v. into syngeneic mice. Mice were killed after 21 d. Lung sections were subjected to immunofluorescent staining of GFP (tumor cells) and PECAM-1 (endothelial cells), followed by confocal analysis and 3D reconstruction. (C) Design of the tail-vein cDNA screen. After transduction with the 4T1 libraries, the 4TO7 cells are injected i.v. in syngeneic mice. Candidate mediators of metastasis are recovered from lung metastases.

Fig. 2.

Design of the gain-of-function cDNA library screen. (A) The flowchart describes the throughput and theoretical redundancy of the cDNA screen. (B) 4TO7-TGL cells stably transduced with empty vector or TM4SF1 were inoculated i.v. into syngeneic mice. Lung colonization was measured by bioluminescent imaging. The panels show representative images (Left), and the graph shows the normalized photon flux at the indicated times (Right). Note that the scale for normalized photon flux is logarithmic.

Fig. 3.

Gain-of-function microRNA library screen. (A) The flowchart describes the throughput and theoretical redundancy of the microRNA screen. (B) 4TO7-TGL cells stably transduced with empty vector or the indicated microRNAs (miR-138, Left; miR-346, Right) were inoculated i.v. into syngeneic mice. Lung metastasis was measured by bioluminescent imaging. The panels show representative images (Top), and the graphs show the normalized photon flux at the indicated times (Bottom). Note that the end time point is 8 wk for miR-138 and relative controls and 6 wk for miR-346 and relative controls.

Fig. 4.

Loss-of-function shRNA library screen. (A) The flowchart describes the throughput and theoretical redundancy of the shRNA screen. (B) ErbB2-TGL cells stably transduced with empty vector or the indicated genes (Numb, Left; Smyd5, Right) were inoculated i.v. into syngeneic mice. Lung metastasis was measured by bioluminescent imaging. The panels show representative images (Top), and the graphs show the normalized photon flux at the indicated times (Bottom).