(D-121) Developing high-throughput pancreatic cancer in vitro model using DNA-directed patterning

Introduction:: With a 5-year survival rate of 11%, pancreatic cancer is considered a lethal disease. This is largely due to non-specific symptoms, leading to a late-stage diagnosis of the disease, and limited response of pancreatic cancer to proposed treatments. One of the most important factors affecting the progression of cancer is the interactions between the different components in the cancer tumor microenvironment (TME). Therefore, modeling the pancreatic TME could give us insight into these interactions and help propose new therapeutic strategies to cure the disease. Current options to study TME include animal models and in vitro models. Resource intensity and lack of control in animal models make them unfavorable for studying the TME. Current in vitro models, on the other hand, frequently fall short in presenting either the complexity or the throughput necessary to study the disease. They often do not reproduce the appropriate cell configuration and, ultimately, the cell-cell interactions that exist in vivo. DNA-directed patterning is a method in which several cell types are labeled with different oligonucleotide sequences to create a complex pattern of these cells on a substrate patterned with the complementary oligonucleotides in the desired configuration. This patterning is performed with lithography, which provides a high-throughput method to develop complex organization. We used DNA-directed patterning to create complex pancreatic cancer models. In this way, we can recapitulate the architecture and cell configuration found in the pancreatic TME and take advantage of the high-throughput nature of DNA-directed patterning to develop a high-throughput in vitro model.


Materials and Methods:: To mimic the configuration of the cells in the pancreatic TME, DNA-directed patterning was used for high-throughput patterning of the cells. The first step was to spin-coat positive photoresist on an aldehyde-functionalized glass slide and then selectively expose it to UV. Next, the exposed photoresist was developed, creating a template, and amine-terminated single-stranded oligonucleotides were introduced to exposed areas of the surface and immobilized by reductive amination. The remaining photoresist was then stripped using acetone. Repeating these steps allows for immobilizing different oligonucleotide sequences and thus patterning of different cell types on the surface. In parallel, different cell types were tagged with complementary oligonucleotides conjugated to a cholesterol molecule, allowing spontaneous intercalation of the cholesterol into the membrane for reversible labeling of the cells. Next, these labeled cells were introduced to the surface and attached to the section featuring the appropriate oligonucleotide sequence through Watson-Crick base pairing. Finally, we incorporated extracellular matrix components to create a “2.5D” system (Figure 1a). Using this method, we were able to mimic the configuration of the cells in a way that resembles pancreatic TME by patterning cells to resemble the histological images of pancreatic TME. We patterned cancer cells in a central ring and designated specific regions for fibroblasts, cancer cells, and two different phenotypes of macrophages (M1-like and M2-like) to resemble the proximity of these cells to cancer cells in pancreatic TME (Figure 1b).


Results, Conclusions, and Discussions:: As shown in the figure, cells are immobilized in the desired configuration. Taking advantage of the high-throughput nature of DNA-directed patterning, we were able to create 256 replicates of the pancreatic cancer TME on a single slide (4 chambers, each containing 64 TMEs). After patterning the cells, we investigated the proper media that we could use for the system. This was done by culturing the cells for a week in different base media, then performing a Live/Dead stain using Calcein AM and propidium iodide. Marker expression and functionality of the cells were examined in order to compare their phenotypes with the phenotypes found in the human body. For this purpose, we used immunofluorescence staining and ELISAs to evaluate the phenotypes of the macrophages and fibroblasts. In this study, we were able to mimic the ratio of the M1-like to M2-like cells in different regions of the TME, having more M2-like macrophages closer to cancer cells while having more M1-like cells being further away from cancer cells. This system could be used to study the interaction of different cell types, the impact of different macrophage phenotypes, and the presence and ratio of different phenotypes of macrophages in the near vicinity of the cancer cells on disease progression.


Through the application of DNA-directed patterning, we were able to develop high-throughput pancreatic cancer TMEs with a predefined configuration of incorporated cells. Future directions include introducing T cells to investigate their functionality within this TME. This system could be used to investigate the cell-cell interactions happening in the pancreatic TME and assess proposed drug and treatment methods for pancreatic cancer.


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