J1 - Spatially defined in vitro model of the pancreatic tumor microenvironment to study reciprocal interactions of cancer cells, macrophages, and, fibroblasts

Introduction: : Pancreatic cancer is a lethal malignancy that is usually diagnosed at late stages due to non-specific symptoms. Most of the current therapeutic interventions, including chemotherapy, surgery, and immunotherapy, have been unsuccessful, in part because the interactions between the tumor and the microenvironment lead to poor therapeutic results. Accordingly, to choose or combine treatment strategies for pancreatic cancer, it is necessary to study the pancreatic tumor microenvironment (TME) and the reciprocal interactions between cancer, stromal, and immune cells. A critical aspect of this environment is the spatial configuration of the cells, which could impact how different cell types behave, both on a single-cell level and collectively. For instance, the distance between cancer-associated fibroblasts (CAFs) and tumor cells impacts whether CAFs develop a myofibroblastic (myCAF) or an inflammatory phenotype (iCAF). That is also the case for macrophages, developing pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes. However, current in vitro models cannot modulate the spatial configuration at this level.

Here, we use DNA-directed patterning to develop a high-throughput model of pancreatic TME with the desired spatial configuration of the cells. DNA-directed patterning combines photolithography with sequence-specific hybridization to precisely position cells using oligonucleotide-tagged surfaces and complementary cell labeling. This will allow for modulating the spatial configuration of the cells in the model in a high-throughput manner. Accordingly, it enables us to pattern different cell types present in the pancreatic TME, including tumor cells, fibroblasts, and macrophages, to recapitulate their organization within the pancreatic TME as part of a high-throughput model to study this microenvironment.

Materials and

Methods: : Briefly, S1813, a positive photoresist, was spin coated on an aldehyde-functionalized glass slide, followed by UV exposure through a photomask. Next, the development process was carried out by immersing the slide in MF-321, which dissolved the UV-exposed S1813 and made the aldehyde functional groups accessible in those areas. Then, amine-terminated single-stranded oligonucleotides were drop cast on the slides and immobilized by reductive amination. Finally, acetone was used to wash the remaining photoresist (Figure 1a, i). By repeating these steps and using different oligonucleotide sequences and masks, it is possible to decorate various areas of the slide with specific oligonucleotides (Figure 1a, ii).

To prepare the cells, cholesterol-conjugated oligonucleotides that were complementary to the ones used for patterning the surface were used to label the cells. Next, these labeled cells were introduced to the slide, and each cell type was immobilized on the area decorated with the oligonucleotide complementary to the one the cells are labeled (Figure 1a, iii). Finally, a collagen solution was incorporated to mimic the extracellular matrix in the TME.

To determine a media to support the coculture of the different representative cell types, mouse embryonic fibroblasts (MEF), murine macrophages, and KPC cells (a murine pancreatic cancer cell line) were cultured in various cocktails of RPMI and DMEM. After a seven-day culture, live/dead staining was performed on the cells to calculate the viability percentage of these cells in different media as a measure to choose the media for the system.

Results, Conclusions, and Discussions:: Through the application of DNA-directed patterning, we were able to develop an in vitro model of the pancreatic TME that resembles the spatial configuration of cells in vivo, where pancreatic cancer cells will be in the core and will be surrounded by a stroma wherein CAFs and macrophages play a significant role in the progression of the disease through their reciprocal interaction. By designating two distinct regions for macrophages, we are able to pattern different ratios of M1 and M2 macrophages based on previous studies showing different compositions near the tumor cells and in the normal stroma. In addition to mimicking the architecture of the TME, we were able to pattern 60 replicates of the model on a single slide (4 chambers, each containing 15 TMEs) (Figure 1b), through the high-throughput nature of DNA-directed patterning.

Since different media are recommended for each cell type in the model, their viability was measured after a seven-day culture in different cocktails of DMEM and RMPI. A 1:1 ratio of RPMI and DMEM could ensure more than 90% viability of all the cell types (Figure 1c).

A collagen solution was used to mimic the ECM within the TME. To ensure that the concentration of the collagen solution does not impact the viability of the cells, different concentrations of collagen were tested in our system, and after a seven-day culture, viability was assessed by live/dead staining. The concentration did not negatively impact cell viability, so a 2 mg/ml collagen solution was used as the material to resemble ECM (Figure 1d).

Acknowledgements and/or References (Optional):: [1] Öhlund D, Handly-Santana A, Biffi G, Elyada E, Almeida AS, Ponz-Sarvise M, Corbo V, Oni TE, Hearn SA, Lee EJ, Chio II. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. Journal of Experimental Medicine. 2017 Mar 6;214(3):579-96.
[2] Zwart ES, van Ee T, Affandi AJ, Boyd LN, Rodriguez E, den Haan JM, Farina A, van Grieken NC, Meijer LL, van Kooyk Y, Mebius RE. Spatial immune composition of tumor microenvironment in patients with pancreatic cancer. Cancer Immunology, Immunotherapy. 2023 Dec;72(12):4385-97.
[3] Scheideler OJ, Yang C, Kozminsky M, Mosher KI, Falcón-Banchs R, Ciminelli EC, Bremer AW, Chern SA, Schaffer DV, Sohn LL. Recapitulating complex biological signaling environments using a multiplexed, DNA-patterning approach. Science advances. 2020 Mar 18;6(12):eaay5696.