Director:
Peter
van Zijl, Ph.D. (PI)
Jeff Bulte. Ph.D. (Co-PI)
Brief Discription and findings:
The aim of this work was to develop and express an
artificial MR reporter gene to use for non invasive, switchable and substrate
independent chemical exchange saturation transfer (CEST) imaging, with the
potential of use for frequency dependent labeling of multiple targets.
Eight synthetic oligonucleotides encoding for lysine rich protein (LRP) were
designed and cloned in tandem into a mammalian expression vector (total of 200
lysine residues; 32kDa) co-expressing enhanced green fluorescent protein (EGFP)
under an internal ribosome entry site (IRES) regulation. The vector was
transfected into a 9L rat glioma cell line. Control cells were
transfected with an empty vector expressing only EGFP. After selection,
two stable cell populations were established, namely cells expressing the
artificial LRP and control cells. LRP expression was verified by
immunofluorescence and showed no inhibitory effect on cell proliferation.
To investigate the potential of imaging the contrast gained from LRP, an MR
phantom was created (Fig. 1a). To remove potential contributions from
processes other than selective saturation transfer from amide protons to water,
the effect of RF irradiation at the amide proton frequency (+3.758ppm from
water) was compared with irradiating at -3.758ppm from water. In the
latter case, no MR contrast was observed (Fig. 1b). Figure 1c displays a
map of the difference in signal intensity (SI) between irradiating at these two
frequencies, normalized by SI at the reference frequency. The average
change in MR signal in extract from LRP expressing cells (0.87%, n=5) was
significantly higher than that of control cells (0.24%, n=4). Moreover,
in pixel-by-pixel t-test maps (comparing the set of images saturated at
Dw=±3.758ppm), the pixels in the capillary containing the LRP extract showed a
significant change in MR signal, which was not detected in the control
capillaries (Fig. 1d). In vivo results are shown in Fig. 2.
Figure 1: CEST
imaging of LRP cell extracts: (a)
phantom layout: PLL, 30 kDa, 100µM (1) and 10µM (2); protein extracts from LRP
cells (3), control cells (4), and PBS (5). (b)
Reference image acquired at Dw=-3.758ppm from the water resonance. (c)
Signal intensity (SI) difference map between RF irradiation at Dw=±3.758ppm,
superimposed on reference image. Pixels outside capillaries were excluded. (d)
t-test
map comparing saturation frequencies Dw=±3.758ppm (only pixels with P<0.05
are color-coded). Scale bar, 1mm.
Figure 2: CEST
imaging of 9L glioma expressing LRP (left) and control cells (right) in a rat
brain. CEST contrast is evident in the LRP expressing 9L tumor but not in
the control tumor, confirming cell extract studies.
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