(F-233) Influence of collagen alignment on E-cadherin expression and cell metabolism in pancreatic ductal adenocarcinoma

Collagen scaffolds are composed of a mixture of gelatin methacrylate and collagen I, crosslinked by the Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator, Second Harmonic Generation (SHG) images of pancreatic cancer patient stroma biopsies served as a blueprint to create collagen patterns on the surface. In total, four scaffold conditions were fabricated and categorized into two models. Model 1 consists of 2 patterns mimicking the primary tumor of one patient while model 2 consists of 2 different patterns from the primary tumor of a second patient. To distinguish the patterns, the straightness of the collagen fibers from models 1 and 2 were analyzed using CT-FIRE software. Pancreatic cancer cells, BxPC3s, were seeded on each scaffold and grown for 24 hours prior to staining for E-cadherin. Immunofluorescence staining of E-cadherin was performed by fixation and permeabilization using paraformaldehyde and Triton X, followed by a 1:100μL primary antibody staining overnight and 1:200μL secondary antibody conjugated with AlexaFluor 594 staining the following day. Finally, a DAPI mounting medium was applied and imaged using two-photon fluorescence microscopy. ImageJ was used to quantify E-cadherin and DAPI fluorescence intensity. Phase-contrast imaging was used to perform time-lapse studies of the seeded cells on the scaffolds with images captured every 30 minutes over 24 hours and analyzed for solidity using ImageJ. Two-photon microscopy was used to investigate metabolic activity by probing the lifetime of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) 24 hours after cell seeding. Fluorescence lifetimes were computed by optimizing a two-exponential decay fit to reduce Chi-squared in SPCImage.

Quantitative assessment of fiber metrics revealed that model 2 has significantly straighter fibers compared to patterns in model 1 (Fig. 1A-B). To study the effect of the different in alignment across collagen models, E-cadherin expression was first analyzed. E-cadherin expression was highest in BxPC3 cells when seeded on model 2 (Fig. 1C-D). Therefore, the increase in collagen alignment in model 2 could be the cause of E-cadherin upregulation. Increased expression of E-cadherin is most well-known for its role in regulating the epithelial-mesenchymal transition (EMT) during metastasis and decreasing cell migratory capacity. The NAD(P)H mean lifetime of cells on model 2, presenting higher E-cadherin expression, was significantly increased relative to model 1 (Fig. 1E), indicating a relative shift towards oxidative phosphorylation. This shift has been shown to mitigate EMT and subsequent migration. Furthermore, E-cadherin-mediated intercellular contacts also enhance cell boundary definition. Thus, the increase in E-cadherin in model 2 indicates that the cells are better able to adhere to one another by forming stronger connections with neighboring cells, making them more compact and circular. This is further supported by the trending increase in the solidity and circularity of cell colonies as shown in model 2 (Fig. 1F-G). Surprisingly, these results suggest that collagen fiber realignment in pancreatic cancer patients may play a role in how collectively migrating pancreatic cancer cells navigate their TME, showing that higher-ordered collagen fibers is correlated with higher E-cadherin expression, compact colony morphology, and metabolic shifts towards oxidative phosphorylation.

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