Purpose: Breast cancer is known for its high morbidity and metastatic potency to lung, liver and brain etc. Chemotherapeutics and biomacromolecule drugs remain to be current choice for breast cancer therapy since conventional operative treatment could not completely eradicate the tumor nidus. Tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL), member of tumor necrosis factor superfamily, is a promising protein-based biomacromolecule drug for tumor therapy. It works by activating tumor cells’ death receptors (DR4/DR5) and following caspase involved cell apoptotic pathway with no side effect for normal tissue. However, direct delivery of TRAIL protein confronts multi-barriers in blood circulation and tumor site. Elimination effect of various protease and complicated tumor microenvironment makes it unevenly distributed in tumor tissue thus retarding the general anti-tumor efficacy. Therefore，it’s vital to maintain protein stability in blood while enhance its tumor penetrating ability.
Recently, cell-based bioreactor system is widely reported for anti-tumor drug delivery. Macrophage is naturally independent with endogenous proteolysis, elimination of reticuloendothelial system and immune surveillance. Meanwhile its innate chronic inflammation homing characteristics makes it a potential cellular vector for tumor targeting drug delivery. However, the coercive drug release mission for macrophage vector is against its engulfing nature of cell debris and pathogens. Herein, a macrophage bioreactor secreting recombinant anti-tumor peptide is developed to boost breast cancer therapy by deep penetrating delivery of TRAIL-TAT (Scheme 1).
Methods: Preparation of pTRAIL-TAT transfected macrophage. Complex of dendritic grafted L-lysine and pTRAIL-TAT was prepared by votexing at N/P ratio of 10:1. After sterilizing filtration, the complex was applied on RAW264.7 cells in serum free medium for 1 hour before scraped for further use.
In vitro antitumor efficacy was tested via transwell device. For upper chamber, 50 μL matrigel was applied before RAW264.7 was seeded and transfected. This chamber was then transfer to 4T1 seeded lower chamber to test the ant-tumor effect of macrophage secreting peptide penetrated from upper chamber. The detection for apoptosis was conducted by TdT-mediated dUTP Nick-End Labeling (TUNEL) kit via fluorescent microscope and flow cytometry.
TRAIL protein and mRNA location was tested by immumohistochemical staining of TRAIL domain and fluorescence in situ hybridization test of TRAIL mRNA sequence on serial paraffin sections of tumor tissue acquired from breast cancer bearing mice treated with saline and macrophage bioreactor transfected with random sequence plasmid, pTRAIL and pTRAIL-TAT, respectively.
Results: A novel pTRAIL-TAT plasmid is constructed by addition of cell penetrating peptide TAT coding sequence. In a transwell ex vivo penetration model (Figure 1A), pTRAIL-TAT transfected macrophage from matrigel coated upper chamber showed significant apoptosis induced effect to 4T1 breast cancer cells from lower chamber indicating the penetrating ability and antitumor efficacy of secreting peptide of macrophage bioreactor (Figure 1B). Flow cytometry of cells in lower chamber showed more apoptotic cells in pTRAIL-TAT group than pTRAIL group indicating the TAT domain contributed to penetration and general tumor cell cytotoxicity (Figure 1C). Tissue morphology, immunohistochemical staining of TRAIL protein and TRAIL mRNA sequence in tumor site showed infiltration of transfected macrophages and dispersive TRAIL distribution in tumor tissue (Figure 2). TRAIL-TAT secreting macrophage bioreactor also showed enhanced anti-tumor efficacy in vivo with reduced tumor volume and alieved liver metastatic potential (Figure 3A/B/C). TRAIL protein and downstream activated caspase-3 was detected in deep tumor tissue from TRAIL-TAT group which verifying the correlation between penetration and apoptosis-inducing effect (Figure 3D/E).
Conclusion: Homologous macrophage was developed as tumor homing vector for the delivery of recombinant antitumor TRAIL-TAT peptide. Due to the natural infiltration ability of macrophage and strong penetrating effect of TAT domain, the intra-tumoral distribution and general anti-tumor efficacy of TRAIL-TAT peptide were improved.