Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems

Abstract

Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.

Gold nanoparticle mediated radiation response among key cell components of the tumour microenvironment for the advancement of cancer nanotechnology

“…HeLa has previously been shown to highly express , an RGD-specific integrin, and may express at least 2 others, and 51 . Further, FB cell lines have been shown to have abundant surface expression of integrins, of a particularly high nature 52 . CAFs have largely been shown to express many different RGD-specific integrins on their surface, partially as a means of crosstalk between cancers cells 53 .…”

EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer

“…After 30 min and 2.5 h, spheroids were rinsed once, washed twice for 10 min with complete culture medium and once with PBS, before fixating them in 4% paraformaldehyde (PFA) for 1–2 h at room temperature. After fixation, PFA was diluted to 2% with PBS and stored at 4 °C until collagen embedding for clearing as described before [ 69 ].…”

“…To clear spheroids, a previously described adaptation [ 69 ] of the SeeDB protocol [ 70 ] was employed. Both protocols use increasingly concentrated fructose solutions supplemented with 0.5 % (v/v) α-thioglycerol.…”

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy

“…Binding properties were taken from the designed ankyrin repeat protein (DARPin) Ec1, which binds EpCAM with an affinity (K d ) of 68 pM [ 21 ]. We assumed a tumor tissue containing a cell density of 2.9 × 10 8 cells/mL, representing a tumor with a high cellularity, and a receptor density of 5.4 × 10 5 receptors/cell, which we previously determined for EpCAM on MCF-7 cells [ 22 ]. As indicated above, we thus considered the tissue as a homogenous medium in which cellular structures are not explicitly defined.…”