A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments

Abstract

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s-1 and 2 µm2 ·s-1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

Mimicking the Biology of Engineered Protein and mRNA Nanoparticle Delivery Using a Versatile Microfluidic Platform

“…While several receptors have been targeted on-chip by us and others in previous work using protein-based agents [8,9,24,25], we report, for the first time, successful targeting of the well-established tumor target CAIX on-chip by CAIX-specific affibodies. For the validation of CAIX targeting, we employed renal carcinoma cell lines that overexpress CAIX, reflecting the CAIX induction upon VHL mutation as widely seen in renal carcinomas [13].…”

“…Based on previous work in which we showed targeted protein delivery using another non-antibody protein scaffold in a similar configuration of the microfluidic device, we selected 1 h as a fixed timepoint. After 1 h, we expected that the affibodies would have diffused through the matrix and reached all cells [9]. We observed a clear membrane staining on the surface of the SK-RC-52 CAIX-positive tumor cells throughout the collagen matrix and no binding to the membrane on CAIX-negative SK-RC-17 cells (Figure 1C and Supplementary Videos S1 and S2).…”

“…A previously published design of the microfluidic chip was used [9]. Briefly, the device, consisting of three parallel channels connected by narrow microchannels (for chip dimensions, see Figure 1B), was fabricated utilizing replica-molded polydimethylsiloxane (PDMS; Dow Corning, Midland, MI, USA) and bonded to a 1 mm thick, 75 × 25 mm plain glass microscopy coverslip or to SuperFrost Plus glass slides with 75 × 25 mm and 175 ± 5 µm thickness (Menzel Gläser, Braunschweig, Germany).…”

“…Through compartmentalization inherent in the design, we were able to add the affibody solutions to the blood vessel-mimicking side channels. By diffusion through narrow channels connecting the side and middle compartments, which mimics the process of extravasation through a leaky endothelium [9], we were able to subsequently image the enrichment at the plasma membranes of cells incorporated as a solid 3D tumor mass after diffusion through the collagen matrix in the middle compartment.…”

“…Hence, organ-on-achip systems have come to the foreground as a complementary approach to study disease and effects of therapeutic agents; in particular, to study transport phenomena [4,6,7]. In our research, we have recently implemented organ-on-a-chip technology for the investigation of drug delivery in various contexts and reported novel insights for retargeted adenoviral vectors and therapeutic proteins targeting various cell surface receptors overexpressed on tumor tissue [8,9]. Notably, we were able to link experimental observations to predictions made by mathematical modeling of drug delivery, ultimately guiding a rational design of protein therapies [10].…”