TY - JOUR
T1 - Targeting carcinoma-associated fibroblasts within the tumor stroma with a fibroblast activation protein-activated prodrug
AU - Brennen, W. Nathaniel
AU - Rosen, D. Marc
AU - Wang, Hao
AU - Isaacs, John T.
AU - Denmeade, Samuel R.
N1 - Funding Information:
NIH grant (5RO1CA124764) and Susan B Komen Grant (to SRD), NCI Prostate SPORE grant (P50CA58236 to SRD and JTI), and DOD prostate cancer predoctoral mentorship grant (W81XWH-07 to WNB). The funders did not have any involvement in the design of the study; the collection, analysis, and interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.
PY - 2012/9/5
Y1 - 2012/9/5
N2 - 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 = 911 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: IC50 = 3.5nM). 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 = 0.206mm 3, 95% CI = 0.068 to 0.344mm3; control: mean = 0.580mm3, 95% CI = 0.267 to 0.893mm3) and 0.24 (treated: mean = 0.131mm3, 95% CI = 0.09 to 0.180mm3; control: mean = 0.543mm3, 95% CI = 0.173 to 0.913mm3), 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.
AB - 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 = 911 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: IC50 = 3.5nM). 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 = 0.206mm 3, 95% CI = 0.068 to 0.344mm3; control: mean = 0.580mm3, 95% CI = 0.267 to 0.893mm3) and 0.24 (treated: mean = 0.131mm3, 95% CI = 0.09 to 0.180mm3; control: mean = 0.543mm3, 95% CI = 0.173 to 0.913mm3), 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.
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U2 - 10.1093/jnci/djs336
DO - 10.1093/jnci/djs336
M3 - Article
C2 - 22911669
AN - SCOPUS:84866096019
VL - 104
SP - 1320
EP - 1334
JO - Journal of the National Cancer Institute
JF - Journal of the National Cancer Institute
SN - 0027-8874
IS - 17
ER -