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Published on: April 28, 2021
Evaluation of an 18F-labeled oligonucleotide probe targeting p21(WAF1) transcriptional changes in human tumor cells
I Koslowsky1, S Shahhosseini, R Mirzayans
1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada.
Abstract:
The ability to image gene expression using 18F-labeled antisense oligonucleotides (asODNs) directed to specific mRNA transcripts during, or immediately following, radio- or chemotherapy would be a valuable clinical tool to monitor the early tumor response to treatment. Imaging of upregulated p21 mRNA postirradiation using 18F-labeled asODNs could offer insights into early tumor responses by detecting signs of accelerated cellular senescence. Thus, the aim of this work was to evaluate the uptake and distribution of a (radio)-fluorinated asODN in vitro in HCT116 p21(+/+) human colon carcinoma cells, asODN and a random sequence oligonucleotide (rsODN) were conjugated with a (radio)fluorine prosthetic group. Irradiated HCT116 cells were treated with naked or liposome-transfected ODNs. Cell fractionation, confocal microscopy, immunofluorescence, and Western blot studies were performed to observe uptake, distribution, and antisense activity of the probes. [F]asODN demonstrated similar antisense binding ability as the unlabeled asODN to p21 mRNA. Liposomal-transfected 18F-labeled asODNs and rsODNs exhibited a three-to fivefold increase in uptake at 2.5 h compared to the naked [18F]ODNs. Distribution of transfected [18F]asODN in the cytoplasm and endosomes increased over time whereas no change in intracellular distribution was observed with transfected [18F]rsODN or naked ODNs. Antisense activity was not compromised with the addition of a fluorine moiety on asODN. The cellular accumulation and distribution of the (radio)fluorinated ODNs was not altered by the addition of the prosthetic group. Radiolabeled ODNs were able to penetrate the cell with preferential uptake observed with the liposome-transfected probes. Increased distribution of [18F]asODN in the cytoplasm suggests the probe is available for targeting its transcript mRNA. This warrants further investigations into the potential of [18F]asODN to image accelerated senescence postirradiation.
Insights
This study developed 18F-labeled antisense oligonucleotides (asODNs) to image gene expression, showing they effectively target p21 mRNA in cancer cells. Liposome delivery enhanced uptake, suggesting potential for monitoring early tumor response to radiation therapy.
Area of Science:
- Molecular Imaging
- Radiochemistry
- Cancer Biology
Background:
- Monitoring early tumor response to radio- or chemotherapy is crucial for effective cancer treatment.
- Imaging gene expression using 18F-labeled antisense oligonucleotides (asODNs) could provide insights into treatment efficacy.
- Upregulation of p21 mRNA post-irradiation is a marker for accelerated cellular senescence, indicating early tumor response.
Purpose of the Study:
- To evaluate the in vitro uptake and distribution of a novel (radio)-fluorinated asODN targeting p21 mRNA in HCT116 human colon carcinoma cells.
- To assess the antisense activity and cellular localization of the radiolabeled probe.
- To investigate the effect of liposomal delivery on the probe's cellular penetration and distribution.
Main Methods:
- Conjugation of a (radio)fluorine prosthetic group to asODN and a random sequence oligonucleotide (rsODN).
- Treatment of irradiated HCT116 cells with naked or liposome-transfected ODNs.
- Cellular uptake and distribution analysis using cell fractionation, confocal microscopy, immunofluorescence, and Western blot.
Main Results:
- The 18F-labeled asODN ([F]asODN) exhibited comparable antisense binding to p21 mRNA as unlabeled asODN.
- Liposomal delivery significantly increased the uptake of [18F]ODNs (asODN and rsODN) by three- to fivefold compared to naked [18F]ODNs.
- Transfected [18F]asODN showed increased cytoplasmic and endosomal distribution over time, suggesting availability for mRNA targeting, while [18F]rsODN and naked ODNs did not exhibit altered distribution.
Conclusions:
- The addition of a fluorine moiety did not compromise the antisense activity or cellular accumulation/distribution of the ODNs.
- Radiolabeled ODNs, particularly when delivered via liposomes, effectively penetrate cancer cells.
- The observed cytoplasmic distribution of [18F]asODN supports its potential for imaging accelerated cellular senescence following irradiation, warranting further investigation.
