We here profiled the transcriptional changes imparted by knockdown of Tlr7 in spheroids formed by the murine breast cancer cell line 4T1. Knockdown of Tlr7 in 4T1 spheroids led to downregulation of genes implicated in cancer cell invasion, proliferation, and metastasis. Overall design: We established spheroids from 4T1 cells transduced either with non-targeting control shRNA or an shRNA leading to knockdown of Tlr7. Subsequently, transcriptional profiling was performed.