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Cell Biology JoVE (Open Access) Citable · DOI

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

DOI: 10.3791/52801-v
What you'll learn
  • ✓ Isolate and expand pancreatic cancer stem cells using sphere culture techniques
  • ✓ Analyze CSC metabolic characteristics including ROS production and oxygen consumption
  • ✓ Apply pharmacological targeting strategies to pancreatic CSCs in vitro
Protocol

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Difficulty
advanced
Total time
~5–7 days (including sphere formation and metabolic assays)
Model organism
Human pancreatic ductal adenocarcinoma cells
Biosafety
BSL-2

Steps

1
Isolate cancer stem cells from pancreatic tumors

Extract and prepare pancreatic ductal adenocarcinoma tissue to enrich for cancer stem cell populations prior to culture.

▶ 01:28
2
Perform sphere formation assay with drug treatment

Culture isolated CSCs in anchorage-independent conditions to form spheres and apply metformin or other therapeutic agents to assess inhibition of sphere formation.

▶ 02:58
3
Quantify ROS production and oxygen consumption

Measure metabolic parameters including reactive oxygen species generation and mitochondrial respiration in treated and control pancreatic CSC populations.

▶ 04:21
4
Interpret metformin effects on CSC function

Analyze and report quantitative results demonstrating how metformin modulates CSC metabolism, sphere-forming capacity, and viability.

▶ 06:09
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