Figure 7. Preclinical testing of drug efficacy against dormant cancer cells.

(a) Gene set enrichment analysis of genes associated with melphalan sensitivity downregulated in the GFP⁺DiD^hi as compared with GFP⁺DiD^neg cells. (b) FACS analysis of total GFP⁺ cells (left panels. % of bone marrow cells) and GFP⁺DiD^hi cells (right panels, % of GFP⁺ cells) for two separate experiments (expt 1 and expt 2). Data show mean±s.e.m. and represent 6–10 mice per group. (c) Three-dimensional maximum intensity projection from ex vivo two-photon imaging of bone from vehicle (top panels) and melphalan-treated mice (bottom panels). Scale bar, 20 μm. (d) Data from Imaris generated spots on mosaic tiles of femurs from vehicle and melphalan-treated mice for number of GFP⁺ cells (left panel) and number of GFP⁺DiD⁺ cells (middle panel) per bone. Percent GFP⁺DiD⁺ of GFP⁺ cells is also shown (right panel). Data show mean±s.e.m. and represent six mice per group (e) FACS analysis of GFP⁺ cells (left panels, % of bone marrow cells) and GFP⁺DiD^hi cells (right panels, % of GFP⁺ cells) in melphalan-treated mice to day 28 (melphalan) or recovered from melphalan (melphalan recovery: 1 or 2 weeks) for two separate experiments (expt 2 and expt 3). Data show mean±s.e.m. and represent 6–10 mice per group, unpaired t-test. (f) Mosaic tiled images of intravital two-photon imaging of the same mouse at days 28 when melphalan was ceased, and day 42 after 2 weeks of recovery following melphalan cessation. Middle panel, higher-magnification images of the region in the white box in the same position of the bone at days 28 and 42. At day 28, small green (GFP⁺DiD^neg) colonies and single red (GFP⁺DiD⁺) cells (white circles) are shown more clearly when the green channel is removed from the image (right panels). At day 42, all of the red (GFP⁺DiD⁺) cells have been activated and the green (GFP⁺DiD^neg) colonies expanded, except for a single red cell (arrow). Scale bars, 500 μm (left panel), 100 μm (middle and right panels).