Lorico Lab
Research Interests
Mechanisms of cell-cell communications in cancer with special emphasis on extracellular vesicles (EVs), including their characterization, intracellular pathways, and possible use as therapeutic targets and cancer biomarkers.
Nuclear transport of EV cargo and viruses: Late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, named “spathasome”, where biomaterials associated with EVs are delivered. This interaction is achieved by a complex of three proteins that work together specifically within nuclear envelope invaginations. These are VAP-A, ORP3, and Rab7 (VOR Complex).
Design, synthesis, and evaluation of anti-cancer and anti-viral activity of novel small molecules.
Evaluation of anti-cancer activity of monovalent anti-CD9 antibodies.
Cells expressing VAP-A-GFP and Rab7-RFP were imaged live by confocal microscopy. Arrows indicate Rab7-RFP+ late endosomes (LE) in nuclear envelope invagination (VAP-A-GFP). Cartoon illustrates the direction (arrow) of Rab7+ LE. Nu, nucleoplasm. Scale bar, 5 µm.
Cells expressing VAP-A-GFP (green) were immunolabeled for ORP3 (red) and Rab7 (magenta) then imaged by confocal microscopy. The inset shows the merged panel with a 4.0 µm-long arrow indicating the area subjected to linescan analysis (left). Thick line indicates the position of late endosomes in nuclear envelope invagination where three fluorescent signals overlaid. Nu, nucleoplasm. Scale bars, 5 µm
PRR851 Effect on VOR Complex
Compounds synthesized in our lab have been shown to disrupt the binding of Rab7 to the ORP3-VAP-A complexes and inhibits translocation of EV-containing late endosomes into NEIs and consequently delivery of EV cargo into the nucleus.
[left] The structures of itraconazole (ICZ), ketoconazole, PRR846 (4-(4-Chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one), and PRR851 (2-(butan-2-yl)-4-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one) drugs are displayed. The reactive groups in ICZ are indicated (dashed boxes). PRR846 was produced from 4-chloroaniline treated with triethyl orthoformate p-toluenesulphonic acid, methyl carbazate and sodium methoxide in methanol (reaction i), while PRR851 was generated from the N-alkylation of PRR846 with 2-bromobutane in the presence of sodium carbonate and 18-crown-6, 2-bromobutane in DMSO (reaction ii).
[right] Cells were incubated with DMSO (control), ICZ, PRR851, PRR846 or ketoconazole for 5 hours, solubilized and subjected to immunoisolation (IS) using anti-ORP3 Ab followed by Protein G-coupled magnetic beads. The input (1/50, for Rab7 only) and entire bound fractions were probed for ORP3, VAP-A and Rab7. The ratio of protein immunoreactivities of the indicated pairs was quantified.
Cells were incubated with DMSO (control), itraconazole (ICZ), PRR851, PRR846 or ketoconazole then immunolabeled for Rab7 and SUN2 and staining with filipin. Cells were analyzed by confocal microscopy. Arrows indicate SUN2+ nuclear envelope invagination (NEI, green) and arrowheads point to Rab7 (red) and filipin-labelled cholesterol (cyan) therein. Bar graph shows the percentage of Rab7+ late endosomes in NEI. Scale bars, 5 µm.
Characterization of EVs derived from SW620
EVs were imaged using dSTORM. Upon immunolabelling, the proteins of interest (CD9, CD63, and CD81) were pseudo-colored as indicated. Single-, double-, and triple-positive EVs are shown and quantified (pie chart). Scale bars, 1 µm (overview), 100 nm (inset).
The compounds itraconazole (ICZ) and PRR851 inhibit EV-induced pro-metastatic morphological transformation in cells. SW480 cells were pre-treated with DMSO (control), ICZ, PRR851, or PRR846 for 10 minutes prior to the addition of SW620 cell-derived EVs and then incubated for 5 hours. Cells were stained for actin (green) and nuclei (DAPI, blue). Bar graphs show the percentage of cells harboring a rounded morphology. Arrowheads and asterisks indicate the rounded cell morphology and the membrane blebbing induced by EVs. Scale bars, 20 µm