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An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment
Published on: July 28, 2012
Antisense Bcl-2 oligonucleotide uptake in human transitional cell carcinoma
B J Duggan1, F E Cotter, J D Kelly
1Uro-Oncology Research Group, Cancer Research Centre, Department of Pathology, The Queen's University Belfast, Belfast, UK. b.duggan@gub.ac.uk
Objectives:
Antisense oligonucleotides (AO) downregulate Bcl-2 protein expression in various tumours if good target cell uptake is achieved. In this study, uptake of FITC labelled AO (FITC-AO) directed at Bcl-2 was examined in: (1) the RT4 bladder tumour cell line; (2) normal pig urothelium, and (3) human superficial bladder tumours.
Methods:
In the RT4 cell line, uptake of FITC-AO, FITC-scrambled and FITC-sense oligonucleotides were quantified by flow cytometry at 4-hour intervals over 24 h. Uptake of FITC-AO was assessed in normal pig urothelium by flow cytometry after FITC-AO was infused for 1 h. Uptake of FITC AO was assessed in samples from 14 human superficial bladder tumours which were maintained in an ex vivo model. In samples from 6 tumours, uptake at 4 h was assessed using fluorescence microscopy. In samples from 8 separate tumours uptake every 4 h within the first 24-hour incubation period was assessed by flow cytometry.
Results:
In the RT4 cell line the FITC-AO, FITC-scrambled and FITC-sense oligonucleotide uptake was similar. Disaggregated cells from the normal urothelium of the 3 pigs exhibited 33, 46 and 51% of cells staining positively for FITC-AO as determined by flow cytometry. All 6 tumour samples had detectable intracellular FITC-AO by fluorescence microscopy at 4 h. In the 8 tumours examined over the 24-hour incubation period, there was a range of percentages of positively staining cells. However, most tumours had a monotonic increase in intracellular fluorescence intensity that plateaued 16 h post-infusion.
Conclusion:
Antisense Bcl-2 oligonucleotides were readily taken up by superficial bladder cancer cells but the heterogeneous uptake in tumour samples needs to be considered when assessing the bioavailability of these drugs.
Insights
Antisense Bcl-2 oligonucleotides show good uptake in superficial bladder cancer cells. However, variable tumor cell uptake necessitates careful consideration for drug bioavailability assessments.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Antisense oligonucleotides (AO) are a promising therapeutic strategy for downregulating specific protein expression, such as Bcl-2, in various cancers.
- Effective delivery and cellular uptake of AO are critical for achieving therapeutic efficacy in tumor treatment.
- Bcl-2 protein plays a significant role in cancer cell survival and resistance to apoptosis.
Purpose of the Study:
- To investigate the cellular uptake of fluorescein isothiocyanate-labeled antisense oligonucleotides (FITC-AO) targeting Bcl-2.
- To evaluate FITC-AO uptake in a bladder tumor cell line (RT4), normal pig urothelium, and human superficial bladder tumors.
- To assess the time-dependent cellular uptake and bioavailability of AO in bladder cancer models.
Main Methods:
- Quantification of FITC-AO, FITC-scrambled, and FITC-sense oligonucleotide uptake in the RT4 cell line using flow cytometry over 24 hours.
- Assessment of FITC-AO uptake in normal pig urothelium via flow cytometry after a 1-hour infusion.
- Evaluation of FITC-AO uptake in ex vivo human superficial bladder tumor samples using fluorescence microscopy and flow cytometry over 24 hours.
Main Results:
- Uptake of FITC-AO, FITC-scrambled, and FITC-sense oligonucleotides was similar in the RT4 cell line.
- Normal pig urothelial cells showed positive staining for FITC-AO in 33-51% of cells.
- All tested human bladder tumor samples demonstrated detectable intracellular FITC-AO, with most showing increased fluorescence intensity plateauing at 16 hours post-infusion.
Conclusions:
- Antisense Bcl-2 oligonucleotides are effectively internalized by superficial bladder cancer cells.
- The heterogeneous cellular uptake observed in tumor samples is a crucial factor to consider when evaluating the bioavailability of these antisense drugs.
- Further research is warranted to optimize AO delivery strategies for consistent therapeutic outcomes in bladder cancer.
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