MRI measurements of reporter-mediated increases in transmembrane water exchange enable detection of a gene reporter

Abstract

Non-invasive imaging of gene expression can be used to track implanted cells in vivo but often requires the addition of an exogenous contrast agent that may have limited tissue access1. We show that the urea transporter (UT-B) can be used as a gene reporter, where reporter expression is detected using 1H MRI measurements of UT-B-mediated increases in plasma membrane water exchange. HEK cells transfected with the reporter showed an increased apparent water exchange rate (AXR), which increased in line with UT-B expression. AXR values measured in vivo, in UT-B-expressing HEK cell xenografts, were significantly higher (about twofold, P < 0.0001), compared with non-expressing controls. Fluorescence imaging of a red fluorescent protein (mStrawberry), co-expressed with UT-B showed that UT-B expression correlated in a linear fashion with AXR. Transduction of rat brain cells in situ with a lentiviral vector expressing UT-B resulted in about a twofold increase in AXR at the site of virus injection.

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Figure 1: Imaging transmembrane water-exchange and measurements of water transport by UT-B in cells expressing UT-B at different levels.
Figure 2: MRI of UT-B reporter gene expression in xenografts.
Figure 3: FEXI measurements of UT-B expression in vivo.
Figure 4: MRI of UT-B reporter gene expression in rat brain.

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Acknowledgements

This work was supported by a Cancer Research UK Programme grant to K.M.B. (17242) and by the CRUK-EPSRC Imaging Centre in Cambridge and Manchester (16465). F.S. is in receipt of funding from the Alexander von Humboldt Foundation in the form of a Feodor Lynen Research Fellowship. We thank S. Patrick for the PGK-S-UTB cell line and his help with the UT-B functional assay and D. Honess and D. McIntyre for helpful suggestions and support with the MRI experiments. We thank M. Strzelecki for cell sorting (Flow Cytometry Facility, Cancer Research UK CI), and the Histopathology Facility and the BRU unit (Cancer Research UK CI) for their help. Lentiviral vector, pBOBI27 was a gift from the Verma laboratory, Salk Institute, La Jolla, USA.

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F.S., S.R., and K.M.B. designed the research; F.S., S.R., D.-E.H., P.D., S.M., R.M., A.J.W., E.M., and A.A.N. performed the research; F.S., S.R., and A.A.N. analyzed the data; S.R. generated the virally transduced cell lines and lentiviral particles, R.J.M.F. contributed the brain model; and F.S. and K.M.B. wrote the paper.

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Correspondence to Kevin M Brindle.

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Integrated supplementary information

Supplementary Figure 1 Validation of FEXI measurements in a sucrose phantom.

a, FEXI reference image without the filter gradient and at the minimum mixing time (tm = 0.01 s; b = 39 s/mm2) from the sucrose phantoms. Sucrose concentrations are indicated on the proton image. b, Circular ROIs were drawn around each tube and within the central water compartment. Mean signal intensity from each ROI and the respective AXR fit according to Equation 1 (in the methods section of the main text) are shown. c, Calculated AXR-map showing two-compartment water exchange only in those tubes containing sucrose. AXR decreases with increasing sucrose concentrations. d, Filter efficiency map showing an increase in filter efficiency with increasing sucrose concentrations.

Supplementary Figure 2 Repeatability of FEXI measurements of AXR and filter efficiency in phantoms of varying sucrose concentrations.

a, The apparent exchange rate first increases and then decreases with increasing sucrose concentrations. b, Filter efficiency increases with increasing sucrose concentration, which can be explained by the increased contribution from sucrose-bound protons to the water ADC at increasing sucrose concentrations.

Supplementary Figure 3 Expression of mStrawberry.

Red fluorescence intensity profiles from the different cell lines, confirming low and high expression of mStrawberry, and therefore UT-B, were determined using a FACSAria II from Becton Dickinson (BD Biosciences, San Jose, USA)

Supplementary Figure 4 Correlation of AXR and UT-B mRNA expression (relative to control).

When corrected for background levels of water transport (in HEK 293T and EF1-L-S cells) the increase in AXR correlated with the increase in UT-B expression (Spearman r = 1, p = 0.0083; linear regression with R2 = 0.88 and slope significantly different from zero, p = 0.018), as determined from measurements of UT-B mRNA levels (Fig. 1 j).

Supplementary Figure 5 FEXI experiment on protein solutions.

AXR was determined from images of tubes containing bovine serum albumin (BSA) at concentrations of 4 % and 35 %. No proton exchange was detected using FEXI. a, Reference FEXI image, without the diffusion filter. b, T2-weighted FSE image. AXR fits showed no observable proton exchange at 35 % (c) or 5 % (d) BSA.

Supplementary Figure 6 Immunohistochemistry of xenografts expressing UT-B in comparison to controls.

Immunostaining in three representative xenografts of the EF1-L-S, EF1-S-UTB low and EF1-S-UTB high cell lines confirmed different expression levels or mStrawberry (RFP staining) and showed high cell viability throughout the xenografts.

Supplementary Figure 7 Immunohistochemistry of two slices from transfected rat brain.

RFP staining to detect mStrawberry expression confirmed the spatial extent of the virus transfection. The green coloured rectangle marks the ROI used for quantitative analysis of the extent of RFP staining.

Supplementary Figure 8 AXR measurements of mixed cell populations containing EF1-S-UTB high and EF1-L-S cells.

a, T2-weighted reference image showing a 5 mm axial slice through the cell pellets. Relative amounts of UT-B expressing cells are indicated on the image. b, AXR map of mixed cell populations shows an increase in AXR with increasing concentration of UT-B expressing cells. c, Mean AXR values from repeated experiments. Data shown in c are mean ± SD. Significance was tested using a Mann-Whitney-Wilcoxon test; significance levels: * p ≤ 0.05, *** p ≤ 0.001.

Supplementary Figure 9 Simulations of the FEXI experiment for varying intracellular volume fractions.

a, Increasing the diffusion filter b-value, bf, reduces the ADC’ at tm = 0, as expected. Increasing intracellular volume fraction also leads to a reduction in ADC’. b, Simulation of filter efficiency values at 3 different diffusion filter gradient strengths (bf = 500, 1500, 2500 s/mm2), where bf = 1500 s/mm2 corresponds to the value used in the in vivo and in vitro studies described here. Filter efficiency corresponds directly to dynamic range in the AXR measurements and is optimal for intracellular fractions around 50 % and at high diffusion filter b-values.

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Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Tables 1 and 2, and Supplementary Notes 1–3 (PDF 1760 kb)

3D FEXI video overlaid on proton images of tissue anatomy (AVI 4369 kb)

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Schilling, F., Ros, S., Hu, D. et al. MRI measurements of reporter-mediated increases in transmembrane water exchange enable detection of a gene reporter. Nat Biotechnol 35, 75–80 (2017). https://doi.org/10.1038/nbt.3714

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