Transcript Cell-Based Selection of Aptamers Specific to Cancer Cells

IF: 21.6(2012)
2010, 43(1):48-57
Presented by Y. Zhang
Nov. 18, 2012
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
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
(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
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.
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
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.
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) 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.
(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).
Medley, C. Anal. Chem. 2008, 80, 1067–1072
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
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. Cellsurface 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
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
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
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
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 NRsgc8c (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 FITClabeled anti-CD5 CEMcells and
NB-4 cells as exposure time
increases.
Dead cell percentages of CCRF-CEM cells (target cells) and NB4 cells (control cells) in all experimental conditions before and
after NIR irradiation
Huang, et al. Langmuir, 2008, 24(20):11860-11865
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
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
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)