The broad specificity of β-gal allows diverse molecular substrates to be developed as potential reporter molecules. Here, agents incorporating a 19F reporter moiety reveal β-gal activity in cell suspensions and breast tumor xenografts in mice. Each substrate and product is characterized by a unique chemical shift, so that multiple agents can be detected simultaneously.
OFPNPG is stable in buffer or with wild type cells. When injected directly into a wild type MCF7 tumor no conversion was detected over 30 mins, although the substrate was found to decline slightly, likely due to wash out (). By comparison, the MCF7-lacZ
tumor immediately showed a large product peak. Over the next 30 mins the substrate declined, presumably due to further conversion and some washout, while the product increased slightly. Our first test in vivo
used OFPNPG since it is more reactive than PFONPG and the pKa of the aglycone (6.03) is outside the normal physiological range minimizing potential line broadening in tumors due to heterogeneous pH. The aglycone of PFONPG has pKa= 6.87 and in vivo
spectra showed a broader signal for the aglycone PFONP compared to OFPNP, but not unduly so (), compared with the substrate peaks with linewidths ca. about 250 Hz. Our initial studies in vivo
used NaTFA as an internal chemical shift reference and isoflurane as the anesthetic. It rapidly became apparent that the reporter molecules themselves could serve as references for identification of cleavage product aglycones, and thus NaTFA could be omitted. Isoflurane was well resolved from substrates and products of these β-gal reporters, but was often visible with two resonances at -5 and -11 ppm, relative to NaTFA = 0 ppm, as described previously (23
). In later experiments we used a non-fluorinated injectable anesthetic to avoid potential signal aliasing when using small spectral acquisition windows (e.g.
An ultimate goal is to image β-gal activity and this was previously demonstrated in cell culture (20
), but currently the signal to noise ratio precludes effective MRI in vivo
. In the current study spatial discrimination was based on injection of two spectrally distinct substrates into separate contralateral tumors. Thus, both tumors could be interrogated simultaneously and independently. Alternatively, localized spectroscopy could be applied using a single substrate, but this is technically more challenging and requires gradients. For this study OFPNPG and PFONPG were selected since they have similar 19
F NMR signals and kinetic characteristics. Discrimination of the contralateral lacZ
- and WT-tumors was immediately obvious upon injection ( and ). However, since the hydrolysis rate for OFPNPG was found to be about twice that of PFONPG in MCF7-lacZ
cell cultures, there was concern that a differential response in vivo
might reflect only the difference in uptake/hydrolysis rates for the two substrates. Thus, investigations were also performed in two phases with substrate switching (). In the first phase, OFPNPG showed activity in the lacZ
-tumor whereas PFONPG showed none in the WT-tumor. Upon alternating the substrates for the second phase five hours later, consistent results were observed with conversion of the PFONPG in the lacZ
-tumor. For the group of four mice with wild type-tumors we achieved 100% negative specificity, i.e.
, none showed any substrate hydrolysis. Unexpectedly, some MCF7-lacZ
tumors showed no apparent β-gal activity by 19
F NMR. However, in tumors where histological comparison was available it was reassuring that these tumors also exhibited much less β-gal activity assessed by X-gal staining.
The current approach reveals relative expression in stably transfected tumor xenografts (), but in vivo
application of reporter genes would likely be related to in situ
transfection, where extensive heterogeneity would be expected in terms of cellular expression. The current approach would likely not differentiate widespread low levels of expression versus highly localized intense expression of transgenes. Indeed, differential bystander effects present a critical issue in enzyme activated pro-drug therapy as encountered previously by Corban-Wilhelm et al.
) with respect to cytosine deaminase activation of 5FC.
The current approach was prompted by the report of Louie et al.
), who presented a 1
H MRI approach to detecting β-gal activity, but their substrate required direct intracellular injection. The 19
F NMR agents reveal β-gal activity based on intratumoral injection, which is feasible for in vivo
preclinical investigations. Ultimately, substrates should be developed, which can be delivered systemically (IP, SC or IV). A key requirement is accumulation or trapping of the enzyme activated reporters at the site of activity. For comparison nuclear imaging has been successfully applied to evaluate thymidine kinase based on accumulation of phosphorylated purine nucleosides (3
). To date, we have observed weak signals of the fluorinated agents presented here in tumors following IP administration, but no conversion, which we attribute to rapid product washout. 19
F MRI of xenobiotic metabolism has been reported, albeit usually at low resolution, as for example with 5FU (27
). These studies required 32 mins per image following 200 mg/kg IP in tumor bearing rats. Stegman et al.
) observed conversion of 5FC to 5FU by cytosine deaminase in tumors following IP administration, but required 20 mins to achieve spectra at 7 T following administration of 1 g/kg. Others (29
) resorted to intra tumoral injection of substrate, as we have used here for detection of β-gal activity. Imaging should be more feasible with higher concentration of reporter molecule. In early studies using β-gal enzyme in solution we showed straightforward Michaelis Menten kinetics for these substrates (17
). However, the product aglycones are toxic (19
) and excessive concentrations seem to inhibit enzyme activity (). Enhanced signal to noise can be achieved by using a CF3
moiety in place of a single fluorine atom, but we have shown that the chemical shift difference is much smaller, and although we have imaged conversion in vitro
, it is probably not feasible in vivo
). Higher magnetic fields should also be advantageous.
Proton MRI approaches are likely to be more suitable for MRI, as reported by others using cells transfected to express transferrin or ferritin (30
). In some cases cells were labeled with ferric irons prior to injection into animals (30
), while in other cases contrast developed in tumors during growth of xenografts (31
). We have achieved preliminary results detecting β-gal activity in tumors by 1
H MRI based on intra tumoral injection of the histology stain S-gal together with ferric ammonium citrate (33
H MRI offers prodigious signal, but detection of transgene activity depends on the contrast-to-noise ratio, which may be difficult to interpret in highly heterogeneous tissues. 19
F NMR has a much lower effective SNR, but the lack of background signal aids interpretation; thus, both approaches have merit.
While β-gal exhibits broad specificity it is reassuring to find that there was no activity by either β-galactosidases (G5160 or E801) at the respective optimal pHs towards the glucoside (PFONPGu, ). Previously, we had shown lack of activity towards α-galactosides (19
). We had hoped to verify that the β-D
-galactosides would resist activity of other enzymes. Unfortunately, we have been unable to obtain β-glucosidases, which are active at physiological pH. When G0395 was tested at its optimal pH=4.5 both β-D
-glucosides and β-D
-galactosides were hydrolyzed. Reassuringly no hydrolysis of the β-D
-galactosides has been seen in WT cells or tumors even after many hours.
In summary, these results further demonstrate and validate 19F NMR gene reporter molecules for detection of β-gal activity. We have used lacZ, but other genes (viz. enzymes) such as glucosidases and glucuronidases would be expected to behave similarly. Initial observations with enzymes in solution have been translated to cells in culture and ultimately tumors growing in vivo. The most important result may be not just identification of WT versus stably transfected tumors, but rather the ability to visualize differential expression.