Cell type-specific pharmacological kinase inhibition for cancer chemoprevention

Abstract

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention.

Keywords: Cancer chemoprevention; Epidermal growth factor; Kinase inhibitor; Liver cancer; Nanoparticle.

Figure 1

Myofibroblast-targeting nanoparticles. (A) Schematic presentation of erlotinib-loaded nanoparticles that target PDGFRB-expressing myofibroblasts (PPB-NP-erlotinib). Erlotinib is released after cellular internalization and inhibits intracellular kinase domain of EGFR protein. In the 3D structural modeling, PPB fits the extracellular domain of PDGFRB with close proximity to the site of PDGF ligand binding. (B) Morphology of the nanoparticles in rat plasma over time by TEM imaging. Scale bar indicates 200 nm. (C) Size of nanoparticles in rat plasma over time measured on TEM images (median of 59 nanoparticles at each time point). Boxes represent 75th and 25th percentile, horizontal line is median, and whiskers mark lowest and highest values. Outliers outside 1.5× of inter-quartile range are shown as open circles. (D) Concentration of erlotinib released from the nanoparticles over time in vitro in rat plasma. (E) Cumulative amount (%) of erlotinib released from the nanoparticles over time in vitro in rat plasma. PDGFRB: platelet-derived growth factor receptor-β, PPB: PDGFRB-binding peptide, NP: nanoparticle, GMBS: N--maleimidobutyryl-oxysuccinimide ester, PEG: polyethylene glycol, EGFR: epidermal growth factor receptor, TEM: transmission electron microscopy

Figure 2

Cellular internalization of myofibroblast-targeting nanoparticles. (A) Upper panels show expression of PGDFRB and GAPDH proteins by Western blotting, and lower panels show fluorescence microscope images in human myofibroblast cell lines, TWNT-4 and LX-2, and hepatoma cell line, Hep G2, incubated with FITC-labeled nanoparticles with (PPB-NP-FITC) or without (NP-FITC) PPB for 24 hr. Scale bar indicates 400 μm. (B) Confocal microscope image of internalized nanoparticles in 0.7 μm-thick slice in TWNT-4 cells incubated with PPB-NP-FITC for 24 hr. Scale bar indicates 40 μm. (C) Fluorescence intensity inside TWNT-4 cells treated with PPB-NP-FITC or NP-FITC (randomly chosen 7 cells for each group). Boxes represent 75th and 25th percentile, horizontal line is the median, and whiskers mark lowest and highest values. (D) Western blotting to quantify p-EGFR suppression by PPB-NP-erlotinib at two doses (0.125 mg and 0.0078 mg). Numbers indicate ratios of p-EGFR to total EGFR protein normalized to untreated controls. PDGFRB: platelet-derived growth factor receptor-β, PPB: GAPDH: glyceraldehyde-3-phosphate dehydrogenase, PDGFRB-binding peptide, NP: nanoparticle, FITC: fluorescein isothiocyanate, EGFR: epidermal growth factor receptor.

Figure 3

In vivo biodistribution of myofibroblast-targeting nanoparticles. (A) Fluorescence signals from each organ isolated from carbon tetrachloride-treated mice after tail vein injection of FITC-labeled nanoparticles with (PPB-NP-FITC) or without (NP-FITC) PPB by IVIS imaging. (B) Quantified fluorescence signals from each organ (n=2 per group). Error bars indicate standard error of mean. (C) Immunohistochemistry of PDGFRB in lung tissues from mice intraperitoneally injected with carbon tetrachloride or PBS (control). Scale bar indicates 100 μm. (D) Immunofluorescence images of liver tissues of PPB-NP-FITC-injected mice. Scale bar indicates 50 μm. (E) Immunofluorescence images of three representative peri-sinusoidal cells in liver tissues of PPB-NP-FITC-injected mice. PPB: platelet-derived growth factor receptor-β-binding peptide, NP: nanoparticle, FITC: fluorescein isothiocyanate, PDGFRB: platelet-derived growth factor receptor-β receptor, glial fibrillary acidic protein (GFAP).

Figure 4

In vivo chemopreventive effect of myofibroblast-targeted EGFR inhibition. (A) Immunohistochemistry of GSTP1 protein. Scale bar indicates 200 μm. (B) Number of GSTP1-positive foci per visual field normalized to respective controls according to the treatment groups (n=8 per group). Wilcoxon rank-sum test p-values (Bonferroni-corrected) are shown. Longer and shorter horizontal bars indicate mean and standard error of mean, respectively. (C) Immunohistochemistry of GPC3 protein. Closed arrow head indicates GPC3-positive focus. Scale bar indicates 200 μm. (D) Number of GPC3-positive foci per visual field normalized to respective controls according to the treatment groups (n=8 per group). (E) Immunohistochemistry of p-EGFR protein. Open arrow head indicates p-EGFR-positive cell. Closed arrow head indicates neoplastic foci with hyper-eosinophilic cytoplasm and GSTP1 positivity in serial tissue section as shown in panel A. Scale bar indicates 100 μm. (F) Number of p-EGFR-positive cells normalized to respective controls according to the treatment groups (n=8 per group). (G) Sirius red staining to visualize and quantify fibrotic tissue. Scale bar indicates 200 μm. (H) Collagen proportionate area (CPA) per visual field normalized to respective controls according to the treatment groups (n=8 per group). *Vehicle control for systemic erlotinib administration (i.e., intraperitoneal injection). **Vehicle control for myofibroblast-targeting nanoparticles (i.e., PPB-NP). PPB: platelet-derived growth factor receptor-β-binding peptide, NP: nanoparticle.

Figure 5

Change in hepatocyte totoxicity and body weight after systemic or myofibroblast-targeted delivery of erlotinib. (A) Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining. Scale bar indicates 100 μm. (B) TUNEL-positive area per visual field normalized to respective controls according to the treatment groups (n=8 per group). (C) Body weight of the rats at the time of sacrifice normalized to respective controls according to the treatment groups (n=8 per group). Weight of livers (D), lungs (E), hearts (F), kidneys (G), and spleens (H) from the rats normalized to respective controls according to the treatment groups (n=8 per group). Wilcoxon rank-sum test p-values (Bonferroni-corrected) are shown. Longer and shorter horizontal bars indicate mean and standard error of mean, respectively. *Vehicle control for systemic erlotinib administration (i.e., intraperitoneal injection). **Vehicle control for myofibroblast-targeting nanoparticles (i.e., PPB-NP). PPB: platelet-derived growth factor receptor-β-binding peptide, NP: nanoparticle.