Speaker
Description
Background. Despite advances in cancer diagnosis and therapy in recent years, pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies due to the pancreas's unique microenvironment. Therefore, innovative targeted therapeutic strategies are urgently required. Aptamers and their conjugates offer a promising approach, as these single-stranded oligonucleotides fold into distinct three-dimensional structures that enable high-affinity and selective binding to target molecules. GreenB1 is an integrin β1 (ITGB1) specific aptamer and it is frequently overexpressed in tumor cells. Characterization across diverse model systems and experimental conditions would facilitate a deeper understanding of integrin expression patterns and evaluate GreenB1’s potential as a vector for targeted therapy. The integration of multiple PDAC model systems including cell lines, patient-derived organoids, and organ-on-chip and microfluidic platforms will enable comprehensive evaluation of aptamer and tumour-cell binding capacity under physiologically relevant and dynamically controlled microenvironmental conditions.
Aim. The aim of this study was to characterise the affinity of the GreenB1 aptamer in pancreatic ductal adenocarcinoma (PDAC) using advanced microphysiological organ-on-chip platform, alongside conventional static 2D and organoid models.
Methods. GreenB1 affinity and experimental condition optimisation were first performed in established PDAC cell lines (CAPAN-2, MIA-PaCa) and in PDAC and normal pancreatic organoids derived from patient biopsies under static conditions, using ImageStreamX Mark II (Cytek Biosciences). These optimised parameters were then translated to organ-on-chip microfluidic system, fabricated using 4-channel CellBox Labs chips. CAPAN-2 cells were seeded at a density of 1 × 10⁶ cells/mL, and 50 µL was transferred to the top channel of the chip. Prior to aptamer binding, cells were cultivated for two weeks until full monolayer confluency was achieved under a flow rate of 2 µL/min. GreenB1 binding under dynamic flow was assessed using immunofluorescence (IF), with additional signal amplification strategies applied to enable GreenB1 detection under microfluidic conditions.
Results. Flow cytometry analysis demonstrated that CAPAN cells exhibited the highest affinity for GreenB1 and were therefore selected for downstream optimisation and organ-on-chip experiments. Optimal binding was achieved with 500 nM GreenB1 following one hour of incubation. Direct detection of GreenB1-FAM by IF was limited due to reduced signal intensity under flow. However, implementation of signal amplification methods enabled successful visualisation of GreenB1 binding in both static cultures and within the microfluidic organ-on-chip system.
Conclusion. This study successfully established and translated GreenB1 binding conditions from static cell culture to a microfluidic organ-on-chip model of PDAC. These findings support the further use of PDAC organ-on-chip models as a proof-of-principle platform for the development and assessment of aptamer-based targeted drug delivery strategies.
Acknowledgements. This study was funded by project „ Targeting pancreatic ductal adenocarcinoma-on-a-chip with aptamer-guided extracellular vesicle delivery”, No. lzp-2024/1-0206.