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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session C

Poster J17 - Implications of Gene Expression on Metastatic Tropism of Pancreatic Cancer in an In-Vitro Microfluidic System

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Pancreatic cancer is among the most aggressive malignancies, known for its high propensity to metastasize, particularly to the liver. Despite advancements in cancer research, the mechanisms driving the selective metastasis of pancreatic cancer cells to specific organs remain poorly understood. Elucidating these mechanisms is crucial for developing targeted therapies aimed at preventing or reducing metastasis, thereby improving patient outcomes.

In vivo models have been employed to investigate the differential organ-targeting characteristics of metastatic human pancreatic cancer cell lines. However, these models present limitations, including the inability to precisely control the microenvironment and the inherent complexity of in vivo systems. To address these challenges, we developed a microchip-based metastasis model designed to study the tropism of pancreatic cancer.

Our microchip model incorporates liver-specific cells (THLE-2) alongside pancreatic cancer cells (AsPC-1 and MIA PaCa-2) within a controlled extracellular matrix (ECM) scaffold environment. This setup allows for the detailed investigation of cell-cell and cell-matrix interactions that drive liver-specific metastasis. By leveraging the advantages of microchip technology and qPCR analysis, we aim to gain deeper insights into the metastatic process and identify potential therapeutic targets. This approach not only enhances our understanding of pancreatic cancer metastasis but also paves the way for the development of more effective treatments tailored to prevent liver metastasis.

Materials and

Methods:  The cancer metastasis chip was manufactured using PDMS on a microfabricated SU-8 mold. The chip features five channels, including two ECM scaffold channels, one pancreatic cancer channel, and two target cell channels, to facilitate interactions between pancreatic cancer cells and target tissue cells (Fig. 1). The ECM scaffold channels were filled with collagen hydrogel to separate pancreatic cancer cells and target tissue cells, allowing only media to pass between cells. After gelation of the ECM scaffold, cell culture media was introduced into the pancreatic cancer and target tissue channels to ensure cell viability before seeding. Pancreatic cancer cells (AsPC-2) and target tissue cells (THLE-2 liver epithelial cells and BEAS-2B lung epithelial cells) were then injected into their respective channels. The invasion and interaction of pancreatic cancer cells with the target tissue cells were monitored under a microscope, and images were captured at regular intervals for 6-8 days to analyze cell behavior and metastatic progression. The invasion angles and number of tissue-tropic AsPC-2 cells were calculated using MATLAB.

To analyze gene expression, RNA was extracted from the pancreatic cancer cells and target tissue cells using the Qiagen RNeasy Mini Kit, followed by reverse transcription into cDNA using the QuantiTect Reverse Transcription Kit. Quantitative PCR was performed to analyze the expression levels of genes related to metastatic behavior. Gene expression levels were normalized to the housekeeping gene RPL7, and relative expression levels were calculated using the ΔΔCt method.

Results, Conclusions, and Discussions: The invasion of AsPC-2 pancreatic cancer cells was observed toward both THLE-2 liver epithelial cells and BEAS-2B lung epithelial cells within the cancer metastasis chip (Fig 2). Polar plots in Figure 2b show a more pronounced invasion toward THLE-2 cells. The number of tissue-tropic AsPC-2 cells and those invading at high angles were significantly higher toward THLE-2 cells compared to BEAS-2B cells . Additionally, the elongation length of AsPC-2 cells, both in total and those with more than 20 µm elongation, was greater toward THLE-2 cells. Compared to an acellular side, more AsPC-2 cells invaded at high angles toward THLE-2 cells, confirming their attraction.

We examined gene expression in THLE-2 cells to understand their attraction to AsPC-2 cells. Key chemokines such as CCL2, CCL5, CCL7, CXCL1, and CXCL12 were over ten times higher in liver cells, while CCL3 was about four to five times higher in lung cells (Fig 3). Most tested growth factors, including VEGF, TGF-β, IGF, and FGF, had similar levels in both cell types, although HGF and EGF were five times higher in liver cells. No significant differences were found in the expression of cancer stemness genes and EMT genes. Thus, chemokines likely drive the metastatic preference of pancreatic cancer cells toward liver tissue over lung tissue.

In conclusion, the study demonstrates that AsPC-2 pancreatic cancer cells exhibit a preferential invasion toward THLE-2 liver epithelial cells over BEAS-2B lung epithelial cells within the cancer metastasis chip. Gene expression analysis revealed that key chemokines, particularly CCL2, CCL5, CCL7, CXCL1, and CXCL12, are substantially more prevalent in liver cells, suggesting that these chemokines play a crucial role in driving the metastatic preference of pancreatic cancer cells to liver tissue. In contrast, growth factors and other tested genes did not show significant differences between pancreatic cancer cells co-cultured with liver and lung cells. These findings highlight the importance of chemokine signaling in the metastatic behavior of pancreatic cancer cells and provide valuable insights for developing targeted therapies to inhibit liver metastasis in pancreatic cancer.

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