Targeting carcinoma-associated fibroblasts within the tumor stroma with a fibroblast activation protein-activated prodrug

J Natl Cancer Inst. 2012 Sep 5;104(17):1320-34. doi: 10.1093/jnci/djs336. Epub 2012 Aug 21.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids / pharmacology
  • Animals
  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects
  • Biomarkers, Tumor / metabolism*
  • Breast Neoplasms / drug therapy
  • Breast Neoplasms / metabolism
  • Calcium / metabolism*
  • Cell Line, Tumor
  • Female
  • Gelatinases / drug effects
  • Gelatinases / metabolism*
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Male
  • Membrane Proteins / drug effects
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Inbred BALB C
  • Neoplasms / drug therapy*
  • Neoplasms / metabolism*
  • Prodrugs / chemistry
  • Prodrugs / pharmacology*
  • Prostatic Neoplasms / drug therapy
  • Prostatic Neoplasms / metabolism
  • Serine Endopeptidases / drug effects
  • Serine Endopeptidases / metabolism*
  • Thapsigargin / analogs & derivatives*
  • Thapsigargin / pharmacology
  • Transplantation, Heterologous
  • Urinary Bladder Neoplasms / drug therapy
  • Urinary Bladder Neoplasms / metabolism


  • Amino Acids
  • Antineoplastic Agents
  • Biomarkers, Tumor
  • Membrane Proteins
  • Prodrugs
  • Thapsigargin
  • Serine Endopeptidases
  • fibroblast activation protein alpha
  • Gelatinases
  • Calcium