A Genome-Wide Screen in Mice To Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation

Abstract

Metastatic colonization, whereby a disseminated tumor cell is able to survive and proliferate at a secondary site, involves both tumor cell-intrinsic and -extrinsic factors. To identify tumor cell-extrinsic (microenvironmental) factors that regulate the ability of metastatic tumor cells to effectively colonize a tissue, we performed a genome-wide screen utilizing the experimental metastasis assay on mutant mice. Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary metastatic colonization (15 increased and 19 decreased; P < 0.005 and genotype effect <-55 or >+55). While several of these genes have known roles in immune system regulation (Bach2, Cyba, Cybb, Cybc1, Id2, Igh-6, Irf1, Irf7, Ncf1, Ncf2, Ncf4 and Pik3cg) most are involved in a disparate range of biological processes, ranging from ubiquitination (Herc1) to diphthamide synthesis (Dph6) to Rho GTPase-activation (Arhgap30 and Fgd4), with no previous reports of a role in the regulation of metastasis. Thus, we have identified numerous novel regulators of pulmonary metastatic colonization, which may represent potential therapeutic targets.

Keywords: B16-F10; lung; metastasis; metastatic colonisation; microenvironment; mouse; mutant.

Figure 1

The metastatic colonization assay. (A) Representative macroscopic image of lungs from wildtype (+/+) and mutant (Tbc1d22a^tm1b/tm1b and Rnf10^tm1b/tm1b) mice 10 days after tail vein dosing with B16-F10 melanoma cells, demonstrating examples of decreased and increased metastatic colonization, respectively. (B) Schematic of the B16-F10 pulmonary metastasis screen, showing that a cohort of mice consists of wildtype mice and groups of different mutant mice, all of which are tail vein dosed with the B16-F10 melanoma cells, and then the number of pulmonary metastatic colonies counted 10 days later (the ‘metastatic ratio’ of a mutant line is derived by dividing the average of the metastases for a mutant group by the average number of metastases for the wildtype group).

Figure 2

Gene Ontology annotation of the 1,300 mutant mouse lines screened as detailed in Methods. A) Molecular functions of genes screened and B) Biological processes of genes screened.