Genetically Engineered Microvesicles Carrying Suicide mRNA/Protein Inhibit Schwannoma Tumor Growth

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein–cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)–an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived “liposomes” to effectively deliver therapeutic mRNA/proteins to treatment of diseases

Macropinocytosis and caveola-dependent endocytosis are major cell uptake pathways for the lysyl oxidase propeptide

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact [email protected] (DScD) --Boston University, Henry M. Goldman School of Dental Medicine, 2015 (Department of Molecular and Cell Biology).Includes bibliographic references: leaves 94-117.Introduction: The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of recombinant rat lysyl oxidase propeptide (rLOX-PP) have been identified , rLOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for rLOX-PP is PI3Kdependent macropinocytosis in PWR-IE , PC3, SCC9 and MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that rLOX-PP buffering capacity recovers PI3K-dependent macropinocytosis in low pH endosomes and facilitates rLOX-PP endosomal escape into the cytoplasm enabling its observed interactions with cytoplasmic targets and ultimately its nuclear uptake. rLOX-PP-Atto565 uptake. MC3T3-1E , PWR-lE, DU145 , PC3, SCC9, Cal27, MDA-MB-23l and MDA-kb2 cell cultures were, respectively incubated with rLOX-PP-Atto565 as a function of time and subjected to confocal microscopy and flow cytometry in the presence or absence of uptake inhibitors. To determine optimum time points for analysis of intact rLOX-PP uptake , we determined the stability of rLOX-PP as a function of time. For this purpose , we generated double-labeled rLOX-PP by sequential labeling rLOX-PP with Atto565 followed by a quencher tag QSY® 9 resulting in a non-fluorescent intact rLOX-PP that would become fluorescent after hydrolysis resulting from separation of the quencher and Atto565. To quench extracellular Atto565 fluorescence, N-[epsilon]- (carboxymethyl)-lysine-bovine serum albumin (CML-BSA) was labeled with QSY® 9 to generate a high plasma membrane affinity, Forster resonance energy transfer (FRET) quencher. To measure endosomal pH change before and after rLOX-PP-Atto565 uptake by cells, a pH sensor , Lysosensor Yellow/Blue dextran 10,000 MW, co-localized with rLOX-PP-Atto565. Fluorescence intensity of punctate images was analyzed using Image J and the fluorescent intensity ratio of 4 70 nm to 525 nm was calculated. All uptake studies were performed in live cells except rLOX-PP-Atto565 and F-actin co-localization studies. Results: Data demonstrated that rLOX-PP-Atto565 enters cells primarily by macropinocytosis in all cell lines. Unlike other cell lines tested, rLOX-PPAtto565 uptake in DU145 cells may occur by a PI3K-independent form of macropinocytosis. Additional LOX-PP uptake pathways also occurred to varying degrees in other cell lines. Our inhibition experiments with 1.5 [mu]M Filipin III in PWR-lE, PC3, SCC9 cells suggest that a caveolae-mediated uptake pathway for rLOXPP-Atto565 occurs. While the major rLOX-PPAtto565 uptake pathway in PC3 cells is by macropinocytosis, these cells also employ a dynamin-and clathrin dependent uptake mechanism for rLOX-PP-Atto565 internalization. In summary, the major uptake pathway for labeled rLOX-PP appears to be primarily PI3Kv dependent macropinocytosis. Secondary pathways appeared to be dynamin- and caveola dependent for labeled rLOX-PP uptake by PWR-3, PC3, SCC9, MDA-MB-231 cell lines. Additionally, it appeared that the elevation of the cationic charge of labeled rLOX-PP mayhelp to drive labeled rLOX-PP into low pH environments. Conclusions: The knowledge that rLOX-PP-Atto565 can be taken up by cells via different pathways and that these pathways vary may provide opportunities to target cells in a specific environment that utilize a relatively rare uptake pathways. Both macropinocytosis- and caveola dependent uptake pathways are favorable for escape from endosomes to the cytoplasm and escape from lysosomal degradation. Furthermore, the endosomal alkalinizing effect of rLOX-PP demonstrated may restore Rae I or PI3K dependent macropinocytosis in cells with a low intracellular pH. This property may increase the uptake of rLOX-PP in tumors compared to normal tissues in vivo. This notion is based on the excess production of lactic acid under hypoxic conditions in tumors resulting in relatively low pH(i)