Selective Targeting of Tumor and Stromal Cells By a Nanocarrier System Displaying Lipidated Cathepsin B Inhibitor

Cathepsin B (CtsB) is a lysosomal cysteine proteinase that is specifically translocated to the extracellular milieu during cancer progression. The development of a lipidated CtsB inhibitor incorporated into the envelope of a liposomal nanocarrier (LNC-NS-629) is described. Ex vivo and in vivo studies confirmed selective targeting and internalization of LNC-NS-629 by tumor and stromal cells, thus validating CtsB targeting as a highly promising approach to cancer diagnosis and treatment.

Labeling of membrane-binding cathepsin B with biotinylated inhibitor. PyMT cells were plated on six-well plate and grown to confluency. Medium was removed, and cells were incubated for 1 hour at 4˚C with ice-cold cell medium containing 50 mM HEPES supplemented with 500 nM of NS-196, the biotinylated form of NS-134. Cells were thoroughly washed and harvested by scraping and lysed on ice in 250 mM Tris-HCl (pH 6.8) and 0.1% Triton X-100 containing 10 mM EDTA for 30 minutes. After clearing by centrifugation at 10,000 rpm g for 10 minutes at 4°C, the supernatants were used as cell lysates. Protein content was determined with DC Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA), and lysates were normalized to equal amounts of protein. Proteins were separated by SDS-PAGE, blotted onto a polyvinylidene difluoride (PVDF) membrane, and detected with streptavidin peroxidase.

Cell cultures preparation.
Primary PyMT cells and MEFs were prepared as described before [2] . Mouse bone marrow-derived macrophages (BMMs) were isolated from bone marrow of a 12-week old male mice FVB strain. Isolated BMMs were differentiated into alternatively activated (or M2) macrophages by incubation with 15% L929 conditioned medium in DMEM medium containing 20% heat inactivated FBS, 1%PS and 1% Glutamax, as a source of M-CSF, for one week.

Proteomic analysis of PyMT and dBMMs conditioned media. The conditioned media of primary
PyMT tumor cells and differentiated BMMs were generated by 24 hours incubation of cells in serum free medium. The obtained conditioned media were then concentrated 10-fold using Centricon filter units with 3000 Da cut-off (Millipore). Samples were separated on a 12.5 % precast SDS-PAGE gel (Lonza) and visualized by silver staining. Each protein lane was cut into six slices and destained. Gel slices were subjected to reduction with 10 mM DTT in 25 mM ammonium bicarbonate, followed by alkylation with 55 mM iodoacetamide in the same buffer. Gel pieces were washed twice with 25 mM ammonium bicarbonate, dried on a Speedvac and rehydrated in 25 mM ammonium bicarbonate containing 1 µg of porcine sequence grade modified trypsin (Promega). Samples were digested overnight at 37°C. Digested peptides were extracted from the gel with 50 % acetonitrile solution containing 5 % formic acid and concentrated to 15 µl. Samples were analyzed with an LTQ Orbitrap Velos (Thermo Scientific) mass spectrometer coupled to a Proxeon-nanoLC (Proxeon) liquid chromatography unit. Peptides were loaded on a C 18 EASY trapping column (Proxeon) and separated on a C 18 PicoFrit ™ AQUASIL analytical column (New Objective) at a flow rate of 300 nL/min. Elution was performed with a 60 minute acetonitrile gradient from 5-40% in 0.1% solution of formic acid, at a flow rate of 300 nL/min. Nine most intense precursor ions in each full scan were selected for CID fragmentation. Dynamic exclusion was set at a repeat count of 1 with exclusion duration of 60 s.
Database searches were performed against IPI mouse database using the MaxQuant software [3] .
Carbamidomethylation of cysteines was set as a fixed and oxidation of methionines as a dynamic modification. Relative quantification of identified proteins was performed using spectral counting with at least 2-fold difference in spectral count being considered as significantly different.