Targeting carcinoma-associated fibroblasts within the tumor stroma with a fibroblast activation protein-activated prodrug.
BACKGROUND
Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG.


METHODS
The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9-11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided.


RESULTS
FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC(50) = 3.5 nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206 mm(3), 95% CI = 0.068 to 0.344 mm(3); control: mean = 0.580 mm(3), 95% CI = 0.267 to 0.893 mm(3)) and 0.24 (treated: mean = 0.131 mm(3), 95% CI = 0.09 to 0.180 mm(3); control: mean = 0.543 mm(3), 95% CI = 0.173 to 0.913 mm(3)), respectively, on day 21 after therapy.


CONCLUSIONS
This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, Baltimore, MD, USA.
W Nathaniel
WN
Brennen
D Marc
DM
Rosen
Hao
H
Wang
John T
JT
Isaacs
Samuel R
SR
Denmeade
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D010450:Endopeptidases; D005260:Female; D018093:Gelatinases / Q000187:drug effects / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000378:metabolism; D012697:Serine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D019284:Thapsigargin / Q000031:analogs & derivatives* / Q000494:pharmacology; D014183:Transplantation, Heterologous; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000378:metabolism; D000099167:Fibroblast Activation Protein Alpha
D000596:Amino Acids; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D002118:Calcium; D010450:Endopeptidases; D018093:Gelatinases; D008565:Membrane Proteins; D011355:Prodrugs; D012697:Serine Endopeptidases; D019284:Thapsigargin; D000099167:Fibroblast Activation Protein Alpha