Aptamers in Cancer Cell Detection and Treatment

1. IF: 21.6(2012) 2010, 43(1):48-57 Presented by Y. Zhang Nov. 18, 2012

2. Introduction Aptamers, "chemical antibodies", antibody-like molecules, function primarily in molecular recognition; Single-stranded oligonucleotides Generated from SELEX(systematic evolution of ligands by exponetial enrichment) Start with a random libray of 1013-1016 ssDNA or RNA Quick and reproducible synthesis Easy and controllable modification to fulfill different diagnostic and therapeutic purposes Long-term stability as dry powder or in solution Ability to sustain reversible denaturation Nontoxicity and lack of immunogenicity Fast tissue penetration

3. Cell-Based Selection of Aptamers Specific to Cancer Cells Cancer-related proteins, such as PDGF, VEGF, HER3, NFkB, tenascin-C, or PMSA Cell-SELEX: proteins may keep their native conformations on cell surface Unnecessary knowing the number or types of proteins on the cell membrane A panel of aptamer probes can be selected to profile the molecular characteristics of the target cancer type

4. (A) Schematics of the cell-based aptamer selection (B)Flow cytometry assay to monitor the binding of selected pools with CCRF-CEM cells (target cells) and Ramos cells (negative cells) CCRF-CEM: cultured precursor T cell acute lymphoblastic leukemia (ALL) cell line Ramos: B-cell line from human Burkitt’s lymphoma

5. Secondary structures of a selected aptamer and the truncated one Schematics of the working principles of monovalent and bivalent NA ligands. (a) 15Apt, a monovalent ligand, has constant ON and OFF and diffuses into bulk solution immediately after dissociation from thrombin, resulting in low inhibitory function. (b) In contrast, when linked to 27Apt to form a bivalent ligand, 15Apt can rapidly return to the binding site after dissociation because of molecular diffusion confined by 27Apt that is still bound to thrombin. As a result, the equilibrium of the reaction is shifted to the left side. Real-time monitoring of light scattering generated by the coagulation process in the presence of different monovalent or bivalent NA ligands (Bi-xSs). After coagulation is initiated by adding fibrinogen to each sample, the reaction kinetics varied depending on the ligands. The initial reaction rate of each sample was calculated (scattering signal increase divided by time, cps/sec) and then plotted in the Inset. This result is consistent with the clotting test. As the number of spacers increased, the reaction rate went down and then up (Inset). Results show that Bi-8S is the best design of bivalent NA inhibitor. Kim, Y. et. al Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5664–5669.

6. Aptamer-Based Cancer Cell Detection Molecular Profiling sgc8, sgc3, sgd3: T ALL sgc4, sgd2: AML, T ALL, B ALL Shangguan, D.Clin. Chem. 2007, 53, 1153–1155

7. Aptamer Nanoparticle Conjugation to Enhance Detection Absorption spectrum and TEM image of Au Ag NRs Fluorescence spectrum of fluorescein-labeled aptamers (a) 25 nM aptamer (b) 0.25 nM aptamer (c) NP+0.25 nM aptamer Flow cytometric assay to monitor the binding of sgc8c (2.5 nM) and NR-sgc8c (0.75 nM) with CCRF-CEM cells (target cells) and Ramos cells (control cells) Binding assay of KK1HO8 (50 nM) and NR KK1HO8 conjugates (1.88 nM) toward K-562 cells. ~80 fluorophore-labeled sgc8 aptamers/nanorod 26-fold higher affinity >300-fold higher fluorescence signal Huang, Y. et al, Anal. Chem. 2008, 80, 567–572.

8. colorimetric assay for sensitive cancer cell detection limit: 90 cells Images of ACGNPS with increasing amounts of target (top) and control cells (bottom) (A-D) TEM images of ACGNPs assembled on different regions of the target cell surface. (E) Image of the control cell surface showing no assembly of the ACGNPs (A) Calibration curve illustrating the relationship between the amount of cells and the absorbance intensity at 650 nm for both target cells (black) and control cells (gray). The assay shows a very good dynamic range in addition to excellent sensitivity. (B) Bar graph showing the change in intensity between the target cells and control cells at 650 nm in both cell media (CM) and fetal bovine serum (FBS) for both cell types. The graph also shows the response of a nontargeting aptamer sequence to each cell type (random DNA). (A) Spectra of different sizes of the ACGNPs with target cells to evaluate the red shift based on particle size. (B) The enhancement of the ACGNPs that is a measure of the signal difference between the assay’s response to target cells versus the same amount of control cells. Medley, C. Anal. Chem. 2008, 80, 1067–1072

9. Cancer Cell Enrichment and Detection Two-nanoparticle assay: Aptamer-magnetic nanoparticles for target cell extraction and enrichment Aptamer-fluorescent dye nanopaticles for cell detection Detection time <1 h Joshua Herr. et. al. Anal. Chem. 2006, 78, 2918-2924

10. Microfluidic poly(dimethylsiloxane) (PDMS) >80% capture efficiency with 97% purity for the target cells Image of device attached to syringe pump on confocal microscope (A). The bottom left inlay shows the device, and the top right inlay shows top-down and sideways views with dimensions. Representative images of original mixture of cells before cell capture assay (B) and channel surface after the cell capture assay performed at 154 nL/sec flow rate (C), with target and control cells stained red and green, respectively. Cell-surface density measured over the course of the cell capture experiment showing linear increase in target cells captured over time (D). Target cell capture efficiency decreases with increased fluid flow rate (E). Joseph A. Phillips, Anal Chem. 2009, 81(3): 1033–1039

11. Aptamer-Based Target Therapy Targeted Intracellular Delivery Liposome vesicles or other delivery vector systems Transferrin-Alexa 633 will both bind to the surface and internalize to the endosomal compartment of CCRF-CEM cells Xiao, Z. Chemistry, A Eoropean Journal, 2008, 14, 1769–1775

12. Targeted Chemotherapy FIGURE 6. Distribution of sgc8c-Dox conjugates inside CCRF-CEM cells after incubation with cells for (A) 30 min, (B) 1 h, and (C) 2 h,respectively. From left to right, the fluorescence confocal images were monitored for sgc8c-Dox, transferrin-alexa633, overlay of these two channels, and bright field channel, respectively. Huang, Y. ChemBioChem. 2009, 10, 862–868

13. Targeted Phototherapy Phototherapy reagent: Chlorin e6(Ce 6) FIGURE 7. Cell toxicity assay results for Ramos cells (P < 0.05) after 30 min incubation, followed by irradiation of light for 4h and subsequent growth for 36 h. Mallikaratchy, P.ChemMedChem. 2008, 3, 425–428

14. Absorption spectrum and TEM image of Au Ag NRs Microscopic images of HeLa cells without NRs (A) and those labeled with sgc8c (50 nM) (B), NR-lib (0.25 nM) (C), and NR-sgc8c (0.25 nM) (D). Cells are irradiated with NIR light (808 nm) at 600 mW for 10 min. Dead cells are stained with PI dye and show red fluorescence. (Left) Fluorescence images of HeLa cells. (Right) Optical images of HeLa cells. Comparison of the relative dead cell percentage between FITC- labeled anti-CD5 CEMcells and NB-4 cells as exposure time increases. Dead cell percentages of CCRF-CEM cells (target cells) and NB-4 cells (control cells) in all experimental conditions before and after NIR irradiation Huang, et al. Langmuir, 2008, 24(20):11860-11865

15. Aptamer-Directed Cancer Biomarker Discovery Biomarker discovery: MS, 2D-GE membrane proteins (30%, <5%) 1) aptamers bound cell lysate 2) membrane proteins separation 3) aptamer-protein complex extraction 4) SDS-PAGE separation 5) MS sequencing 6) target protein validation 1, markers; 2, membrane extracts; 3, protein captured with the nonbinding sequence; 4, magnetic beads only; 5, protein captured with sgc3b; 6, protein captured with sgc8c. Shangguan, D. J Proteome Res. 2008, 7(5): 2133–2139

16. Conclusion and Future Perspective Cell-SELEX: aptamer probes Interaction between aptamers and cells New cancer biomarker Cancer research: biochemistry and molecular basis Cancer detection, diagnosis, treatment Molecular profiling of blood or body fluids Personalized medicine

18. Immunity in the tumor microenviroment Regulatory events/networks in the tumor microenviroment Inflammation in the tumor microenvironment Functional genetics of fibroblasts in the tumor microenviroment Cytokine and chemokine networks in the tumor microenviroment Targeting the tumor and the tumor microenviroment VEGF, IL-6, TAK1(TGF-β-activated kinase)