Figure 7.
Fate of Rluc-RFP-TK-FP-hAMSCs Implanted in Tumors Subject to GCV Treatment
Therapeutic Rluc-RFP-TK-FP-hAMSCs were implanted in Pluc-GFP-U87 tumors (8 × 105 Rluc-RFP-TK-FP-hAMSCs, day 6) and treated with i.p. GCV (50 mg/kg, day 10) during a 37-days period and subject to the CLARITY procedure. (A) Pluc-GFP-U87 cells were detectable by non-invasive in vivo BLI after 37 days of treatment. (B–F) Transparent brain slices (500 μm thick) were analyzed by Leica SPE fluorescence confocal microscopy equipped with 20× and 40× dry objectives to visualize fluorescent cells. (B) Tridimensional reconstruction of the therapy surviving green fluorescent Pluc-GFP-U87 tumor after 37 days of bystander therapy (550 × 550 μm, depth: 90 μm). Red fluorescent therapeutic Rluc-RFP-TK-FP-hAMSCs are found in close association with tumor cells. (C) Inset: maximum projection of a therapeutic cell node (red) surrounded by tumor cells (green) (137.5 × 137.5 μm, depth: 24 μm). (D) Inset: maximum projection of an Rluc-RFP-TK-FP-hAMSCs vessel-like structure (delineated by dotted lines and indicated by the arrowhead) containing red blood cells in its lumen (→), surrounded by tumor cells (137.5 × 137.5 μm, depth: 48.9 μm). (E) Orthogonal view of inset (D) showing red fluorescent cells (arrowheads) forming the blood vessel with red blood cells in its lumen (→) and tumor cells surrounding the vessel. (F) Surface intensity tridimensional reconstruction of the Rluc-RFP-TK-FP-hAMSCs vessel-like structure (arrowheads) in inset (D). Animated tridimensional reconstruction of the Rluc-RFP-TK-FP-hAMSCs vessel-like structure is also shown in Video S1.