Figure 5.

Fluorescence Confocal Microscopy Analysis of CLARITY-Processed Mouse Brains

Three mice bearing fluorescent tumors, one intact and two resected tumors (day 21) that had been treated or not with Rluc-RFP-TK-FP-hAMSCs, were subjected to the CLARITY procedure (day 32) to render brain tissue transparent. Cleared brain slices (500 μm) were analyzed by fluorescence confocal microscopy to visualize fluorescent cells. Images were taken with a Leica SPE equipped with 10× and 20× dry objectives. The microscope images shown are maximum projections of multiple Z-scans. (A) Fixed mouse brain prepared for the CLARITY procedure and slicing pattern. (B) Image showing a brain slice before (right) and after (left) CLARITY. (C) Maximum projection of a 3D-stitching composite from a non-resected Pluc-GFP-U87 tumor (2,453 × 1,884 μm, depth: 154.9 μm). (D) Maximum projection of a 3D-stitching composite (2,556 × 2,045 μm, depth: 94.8 μm) from a resected Pluc-GFP-U87 tumor. Resection cavity borders are indicated by a gray line. Residual tumor cells are detected in the cavity (green fluorescence signal). (E and F) Images from two transparent brain slices from a mouse subjected to surgery and bystander therapy. (E) Image of the first slice generated as a 3D-stitching composition from a tissue volume of 1,319 × 2,006 μm, depth: 149.7 μm, showing an incipient and apparently organized Pluc-GFP-U87 tumor of approximately 400-μm diameter and red fluorescent therapeutics cells (→) in apparent migration between the plasma clot (top of the image) and the tumor (inset; magnification of the interaction between therapeutic and tumor cells). Some tumor cells (arrowhead) appear also in apparent migration toward therapeutic cells. (F) Image of the second slice generated by 3D-stitching composition of a tissue volume of 467 × 761 μm, depth: 40 μm, showing an interaction zone of therapeutic and tumor cells.