RNA Analysis of Hypoxia Markers for Pancreatic Cancer Cells Cultured in 3D

This webinar reviews: • 1:38 - 4:18: Introduction to Pancreatic Ductal Adenocarcinoma (PDAC) • 4:19 - 8:23: Spheroid Formation and Culture • 8:24 - 13:17: RNA Isolation and RT-qPCR • 13:18 - 16:05: IF Labeling of Hypoxia Markers • 16:39 - 25:19: Q&A Session Pancreatic ductal adenocarcinoma (PDAC) is a notoriously aggressive tumor type due to its high levels of metastasis and recurrence. Pancreatic tumors typically lack vasculature and are hypoxic due to oxygen diffusion limitations. Hypoxia can lead to the transcriptional induction of a series of genes that participate in angiogenesis, tumor growth, invasion, and metastasis through hypoxia-inducible factor-1 (HIF-1), an oxygen-sensitive transcriptional activator. These genes, which include glucose transporter protein type 1 (GLUT-1) and carbonic anhydrase IX (CA IX), are associated with a decreased survival in PDAC patients. To model PDAC in vitro, three-dimensional (3D) cell culture models can be used as they have been shown to develop gradients of diffusion for oxygen and glucose at diameters >200 µm, and can develop hypoxic cores. We demonstrate this by culturing the PDAC cell line PANC-1 in Corning® spheroid microplates and isolating RNA using the Promega™ Maxwell® RSC instrument. RNA transcript analysis was performed using Promega reagents. Relative expression of hypoxia markers HIF-1α, GLUT-1, and CA IX were analyzed relative to 2D culture using RT-qPCR to assess the correlation between spheroid size and hypoxia marker expression.