This paper explores using a dual-tracer approach and molecular imaging to quantify cell surface receptor expression in live tumor cell cultures. Infrared dyes IRDye 700 and IRDye 800, which binds to epidermal growth factor receptors, are injected into U251 Human Glioma Cells. Imaging shows IRDye 800 absorbed to cells and IRDye 700 remaining in free space. A compartment model relates the concentration of tracers in plasma, free space, and bound regions. Results demonstrate a correlation between dual-tracer measured receptor concentrations and cell number, showing this approach can quantify receptor dynamics during tumor development.

1. Quantifying Cell Surface Receptor Expression In
Live Tissue Culture Media Using Dual-Tracer
Approach
Aparna Singh1 , , Clover Xu1, Lagonojita Sinha1,
Jialing Xiang2 ,Cynthia Yang 1, Ken M Tichauer1
1Armour College of Engineering, Illinois Institute of Technology,
2College of Science, Illinois Institute of Technology
Chicago, IL 60616, USA
A dual-reporter methodology has been used to
identify the uptake of Infrared dyes by U251
Human Glioma Cells. The moderate levels of
Epidermal Growth Factor Receptors expressed by
this cell line can help quantify available receptor
binding potential of tumors in vitro within a
relevant time scale 1.
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Introduction:
• Cancer, or malignant neoplasia, involves an
unregulated growth of cells.
• This often leads to genetically heterogeneous
subpopulations of cells within a single tumor.
• The heterogeneity of tumors makes it very
difficult to treat1.
Purpose: This paper explores the usefulness of
‘dual tracer’ model and molecular imaging to map
uptake of Infrared dyes in U251 Human Glioma
cells and map the molecular expressions of
Epidermal Growth Factors in U251 Human Glioma
cells.
Procedure: This will be studied by injecting two Infrared dyes – IRDye 700 NHS Ester and
IRDye 800 EGF. Due to the presence of EGFR in the tumor line, IRDye 800 will bind to them while
IRDye 700 will remain unbounded to tumor and hence will remain in the “free space”. Later washing
these cell lines and the free space with PBS will help in quantifying the Binding Potential (BP).
Moreover graphing the fluorescence units with respect to washes will identify the amount of dyes that
will be left absorbed by the cells
When there is no targeted binding and
when first order tracer kinetics is taken
into consideration, the following
differential equation can be used to
model relationship between the
concentration of tracer in the plasma
compartment and in the free
compartment2:
Result:
The wells were first imaged after the
dyes were removed from gel after 45
minutes. Following were the IRDye
700 NHS Ester and IRDye 800 EGF
absorption respectively:
Figure 1: IRDye
700 NHS Ester in
the gel plates after
45 minutes
Figure 2: IRDye
800 EGF in the
gel plates after 45
minutes.
When there is a targeted binding, there
is an additional compartment added to
the model. Consequently, the uptake of
the tracer in the bound region:
The concentration of tracer
in the free space will now
have to take into account the
concentration of tracer in and
out of the bound region.
Hence
Dual -Tracer Compartment
Model:
Conclusion: Results from these studies
demonstrated a clear correlation between dual-tracer
measured concentrations of epidermal growth factor
receptor and cell number. This study demonstrated
that a dual-tracer imaging approach could
successfully be employed to quantify the receptor
concentration in U251 Human Glioma Cells. In
addition it has potential to carry out the first repeated
measures of cell surface receptor dynamics during 3D
tumor mass development.
Targeted binding:
References:
1.Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next
generation. Cell 144, 646-674, (2011).
2. Lammertsma, A. A., Bench, C. J., Hume, S. P., Osman, S., Gunn,
K.,Brooks, D. J., and Frackowiak, R. S. J. 1996. Comparison of methods
for analysis of clinical [11C]raclopride studies. J. Cereb. Blood Flow
Metab. 16:42–52.5. Davies MJ, Gordon JL, Gearing AJ, Pigott R, Woolf
N, Katz D, Kyriakopoulos A. The expression of the adhesion molecules
ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J
Pathol. 1993; 171: 223–229.
Untargeted binding:
dCb(t)
dt
= k3Cf (t)−k4Cb(t).......(2)
dCf,x (t)
dt
= K1,xCp,x (t)−k2,xCf,x (t).......(1)
!!!
dCf (t)
dt
=K1Cp(t)−k2Cf (t)−k3Cf (t)+k4Cb(t).......(3)
Acknowledgements:Additional
support for this work comes from an IIT ERIF
award (Tichauer/Xiang)
Mapping molecular heterogeneity in tumors for
advanced personalized cancer therapy
K1,t
k2,t
K1,t
k2,t