22-11-2012, 04:33 PM
Quantitative molecular profiling of biomarkers for pancreatic cancer with
functionalized quantum dots
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Abstract
Applications in nanomedicine, such as diagnostics and targeted therapeutics, rely on the detection and targeting of membrane biomarkers.
In this article we demonstrate absolute quantitative profiling, spatial mapping, and multiplexing of cancer biomarkers using functionalized
quantum dots (QDs). We demonstrate highly selective targeting molecular markers for pancreatic cancer with extremely low levels of
nonspecific binding. We confirm that we have saturated all biomarkers on the cell surface, and, in conjunction with control experiments,
extract absolute quantitative values for the biomarker density in terms of the number of molecules per square micron on the cell surface. We
show that we can obtain quantitative spatial information of biomarker distribution on a single cell, important because tumors' cell
populations are inherently heterogeneous. We validate our quantitative measurements (number of molecules per square micron) using flow
cytometry and demonstrate multiplexed quantitative profiling using color-coded QDs.
Introduction
The detection of cancer biomarkers is important for diagnosis,
disease stage forecasting, and clinical management. As tumor
populations are inherently heterogeneous, a key challenge is the
quantitative profiling of membrane biomarkers, rather than
secreted biomarkers, at the single-cell level. The detection of
cancer biomarkers is also important for imaging and therapeutics,
in that membrane proteins are commonly selected as targets. Many
methods for detection of membrane proteins yield ensemble
averages and hence have limited application for analysis of
heterogeneous populations or single cells. Fluorescence-based
methods allow detection at the single-cell level; however,
photobleaching presents a major limitation in obtaining quantitative
information. Quantum dots (QDs) overcome the limitations
associated with photobleaching; however, realizing quantitative
profiling requires stable quantum yield, monodisperse quantum
dot–antibody (QD-Ab) conjugates, and well-defined surface
chemistry.1 By quantitative profiling we specifically refer to
methods that yield absolute values of expression levels (e.g.,
number per square micron) and not relative values. Here we
demonstrate quantitative profiling, spatial mapping, and quantitative
multiplexing of molecular biomarkers associated with
precursor lesions of pancreatic adenocarcinoma at the single-cell
level using QD-Ab conjugates.
Methods
Synthesis of QDs
Most experiments were performed using CdSe/(Cd,Zn)S core/
shell QDs with an emission wavelength of about 610 nm.32,33 For
multiplexing experiments we synthesized CdSe/(Cd,Zn)S core/
shell QDs with an emission wavelength of 524 nm and CuInSe/
ZnS core/shell QDs with an emission wavelength of 707 nm.
Water solubilization of QDs
Water-soluble QDs were obtained by forming a lipid monolayer
composed of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine/
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lauroyl)–
polyethylene glycol 2000 (MHPC/DPPE-PEG2k; 80:20 mol%) or
MHPC/DPPE-PEG2k/DPPE-PEG2k-COOH (80:15:5 mol%).
Typically 0.25 nmol of QDs, 4 μmol of MHPC, 0.75 μmol of
DPPE-PEG2k, and 0.25 μmol of DPPE-PEG2k-COOH were
dissolved in 0.3 mL of chloroform. This solution was added to
2 mL of deionized water, heated, and maintained at 110°C for
1 hour under vigorous stirring to evaporate chloroform. The
resulting solution was sonicated for 1 hour, then centrifuged, and
the supernatant then passed through a syringe filter with a 200-
nm polytetrafluoroethylene membrane (VWR, Radnor, Pennsylvania)
to remove any aggregates or unsuspended QDs. Quantum
yield measurements were performed on suspensions with about
100 pmol QDs in 4 mL deionized water using a Hamamatsu
C9920-02 fluorometer (Hamamatsu, Hamamatsu City, Japan).
Details are provided in Supplementary Materials.
Imaging
Briefly, about 105 cells (see above for description of cell
lines) were pre-seeded in a 12-well culture dish. At 50–70%
confluency (1–2 days), the cell medium was aspirated and the
cells washed three times with PBS. Fixing solution (3.7% v/v
formaldehyde) was added to the wells for 20 minutes, followed
by washing three times with PBS. The cells were then incubated
with a blocking buffer (10% horse serum or 5% bovine serum
albumin in PBS) for 1 hour before introducing 500 μL of QD-Ab
conjugates to each well and then incubating at room temperature
for 30 minutes. In all profiling experiments, cells were incubated
with 20 pmol QDs, corresponding to a dose of about 108 QDs
per cell. In experiments to confirm that the membrane biomarkers
were saturated with QD-Ab conjugates (see Figure 3,
A), cells were incubated with 0.1–20 pmol QDs. Next, the
QD-Ab solution was aspirated and the cells washed with PBS
three times. The maximum biomarker density (∼500 μm–2)
corresponds to about 105 per cell or a maximum QD excess of
about 1000 QDs per biomarker.
Flow cytometry analysis
Cell were centrifuged at 500 g for 5 minutes and washed three
times in an isotonic PBS buffer supplemented with 0.5% (v/v)
bovine serum albumin to remove contaminating serum components
that may be present in the culture medium. Cells were
resuspended in the same buffer to a final concentration of 4 × 106
cells mL–1 and 25 μL of cells (105 cells) transferred to a test
tube. A 10-μL aliquot of phycoerythrin (PE)-conjugated
aCLDN4 antibodies (IgG2A) was then added to the test tube
and incubated for 30 minutes. As a control for analysis, cells in a
separate tube were treated with a PE-labeled mouse IgG2A
isotype control. See Supplementary Information for details.
Image analysis
Immunofluorescence images were acquired and analyzed
using Nikon NIS-Elements AR 3.1 software. The software was
used to automatically select the cell boundaries and to generate
the pixel statistics of the cellular region. The average
fluorescence intensity per square micron within the cellular
region was determined quantitatively, which allows us to make
quantitative comparisons between different cell lines and
different antibodies (i.e., different molecular biomarkers).
Control experiments included: (1) PEGylated neutral-charge
(zwitterionic) QD-L-PEG (no antibody) incubated with pancreatic
cancer cell lines and a normal pancreas epithelial cell line
(HPDE), and (2) QD-Ab conjugates incubated with HPDE cells.