Figure 1
Migration of CTL007 toward WC007 CRC cells in the reconstruct. A. Reconstruct schema. B. [a-f]: The bottom layer of reconstructs contained 1.8 × 105 fibroblasts in 450 μl type I collagen gel which was followed by addition of CRC cells (1 × 105) on top after 24 hr. After further 24 hr, a separating layer of fibroblasts in collagen gel (100 μl, 500 μm) was added on top of cancer cells, followed by the top layer containing CTL (1 × 105 [32]), or autologous PHA blasts (control lymphocytes [a, c, e]) mixed with 1 × 105 fibroblasts and collagen. Reconstructs were harvested on day 6 (3 days after adding T cells), fixed in buffered formalin and embedded in paraffin. a, b: Staining with H&E. c, d: Specific brown staining of apoptotic cells by anti-caspase 3 Ab. e, f: In situ end-labeling (ISEL); black staining of nuclei of cells undergoing apoptosis. Apoptosis was significantly higher in presence of CTL (d and f) than in presence of autologous PHA blasts (c and e; p < 0.0005). g-h: Reconstructs were prepared as in a-f, but tumor cells were stained with CellTracker Blue CMAC; autologous PHA blasts (g) and CTL007 (h) were pre-stained with CFDA-Green. Reconstructs were harvested on day 4 (2 days after adding T cells), and sections were photographed in the Nikon fluorescence microscope using appropriate filters. Arrows indicate binding of CTL007 to the tumor cells.