Figure 4.
Stromal-selective cell death in tumors treated with FAP-activated prodrugs. A) FAP-positive stromal cells (red) selectively undergo apoptosis as indicated by TUNEL-positive (green) nuclei following treatment with a FAP-activated prodrug, ASGPAGP-A12ADT, whereas untreated control tumors show a more general pattern of cell death in MCF-7 xenografts. Nuclei are counterstained with DAPI (blue). Images are representative of three images per tumor and three tumors per group. Images were taken at both ×10 and ×20 magnifications. Scale bars = 100 μm. B) Stromal cells positive for α-smooth muscle actin (red) selectively undergo apoptosis as indicated by TUNEL-positive (green) nuclei following treatment with a FAP-activated prodrug (ASGPAGP-A12ADT or DSGETGP-A12ADT) compared with untreated control tumors from mice bearing MCF-7 xenografts. Nuclei are counterstained with DAPI (blue). Images are representative of three images per tumor and three tumors per group. Images were taken at ×20 magnification. Scale bars = 100 μm. C) There are approximately 90% more TUNEL-positive cells in tumors treated with a FAP-activated prodrug. Quantitation of images represented in (A). Average of 6–9 images per group. Error bars represent 95% CI. Asterisks indicate statistically significant P < .05, determined using mixed effects models. (Note: the analysis was based on each individual replicate, not their mean.) D) Approximately 80% of the total apoptotic (TUNEL-positive) cells from the FAP-activated prodrug-treated tumors are of stromal lineage compared with approximately 40% of the total TUNEL-positive cells in the untreated control tumors. Quantitation of images represented in (C). Average of nine images per group. Error bars represent 95% CI. Asterisks indicate statistically significant difference from untreated control, with P < .05 determined using mixed effects models (Note: the analysis used each individual replicate.) E) Treatment of tumors with a FAP-activated prodrug produces a bystander effect resulting in the death of cells adjacent to areas of FAP expression. White arrows indicate desmin-positive pericytes (left panel, red) and CD31-positive endothelial cells (right panel, red) in the stromal compartment undergoing apoptosis (TUNEL-positive, green) in FAP-activated prodrug-treated tumors. Nuclei are counterstained with DAPI (blue). Images taken at ×40 magnification. Scale bars = 50 μm. F) Stromal cells within regions of FAP (red) expression in xenografts begin to proliferate (arrows) following treatment with a FAP-activated prodrug (right panel) compared with those in untreated controls (left panel) as indicated by cells staining positive for an anti-mouse Ki-67-specific antibody (green). Nuclei are counterstained with DAPI (blue). Images are representative of six images per tumor and three tumors per group. Both images were taken at ×10 magnification. G) There is approximately fivefold greater proliferation of mouse stromal cells in MCF-7 xenografts treated with a FAP-activated prodrug. Quantitation of images represented in (F). Average of nine images per group. Error bars represent 95% CI. Asterisks indicate statistically significant difference from untreated control, with P < .05, determined using mixed effects models (Note: the analysis used each individual replicate.) TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; FAP = fibroblast activation protein α; RFU = relative fluorescence units; DAPI = 4′,6-diamidino-2-phenylindole; CI = confidence interval.