Figure 2. FASN knockdown in CA1d cells impairs tumor outgrowth and induces a dormant-like phenotype.

(A) FASN depletion inhibits tumor growth in an orthotopic model. Control (CA1d/vector control) and FASN-depleted cells (CA1d/FASN shRNA #28), injected into the fat pad of the mouse mammary gland, were monitored over the course of 7 weeks and tumor growth was assessed twice weekly. Tumor growth from one representative experiment out of three independent experiments, with similar results, is presented as mean ± SD. (B) FASN depletion decreases tumor volume. Distribution of tumor sizes from control and FASN-knockdown cells at 14, 25, and 36 days after injection. Note that only extremely small tumors were observed in FASN-depleted cells. Results are presented as mean ± SD (n = 12); p < 0.001. (C) Representative images of tumors originating from control and FASN-depleted cells in mice 6 weeks post injection. (D) Depletion of FASN restores a non-malignant, dormant-like, phenotype in CA1d cells. Histology of tumors derived from control (CA1d/vector control) and FASN-knockdown (CA1d/FASNshRNA#28) cells. Paraffin-embedded tumor sections were stained for H&E and FASN. Note the nearly normal looking ducts in the FASN-depleted cells. (E) Ducts from tumor-derived FASN-depleted cells are of human origin. Fluorescently labeled human (green) and mouse (red) home-brew whole genomic FISH probes were used to distinguish human and murine cells. (F) Visualization of invasive breast tumor cells by staining with smooth muscle actin (SMA, red) and fatty acid synthase (FASN, green). Note that in FASN-depleted tissues, a SMA-positive monolayer of cells surrounds a hollow lumen, while it is distributed within the tumor in control tissue. (G) FASN depletion reduces cell proliferation and reverses the aggressive tumor phenotype. Note high expression of vimentin, a marker for aggressive invasive tumors in control tumors, but not in tumors from FASN-depleted cells.