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Cell Biology JoVE (Open Access) Citable · DOI

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

DOI: 10.3791/2169-v
What you'll learn
  • ✓ Isolate cancer stem cells from pancreatic cancer xenografts using FACS.
  • ✓ Apply flow cytometry markers (ALDH, CD44, CD24) to identify CSCs.
  • ✓ Generate viable single-cell suspensions from tumor tissue for downstream analysis.
Protocol

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Difficulty
advanced
Total time
~4–6 hours per xenograft (harvest to sorted population)
Model organism
Mouse (human pancreatic adenocarcinoma xenograft)
Biosafety
BSL-2

Steps

1
Introduce cancer stem cell isolation rationale

Review the biological significance of cancer stem cells in malignancies and the experimental strategy for isolating CSCs from pancreatic tumor xenografts using ALDH activity and CD44/CD24 surface markers.

▶ 01:05
2
Harvest pancreatic xenografts from mice

Surgically remove established human pancreatic adenocarcinoma xenografts from mouse hosts following institutional IACUC protocols.

▶ 02:28
3
Prepare single-cell suspension from xenograft tissue

Dissociate intact xenograft tissue into a single-cell suspension using enzymatic digestion and mechanical disaggregation.

▶ 03:22
4
Remove debris and dead cells by density separation

Eliminate tissue debris and non-viable cells via density gradient centrifugation or magnetic bead-based negative selection to enrich viable cell population.

▶ 04:12
5
Stain cells with FACS markers for CSC identification

Incubate cells with fluorescently-labeled antibodies against CD44 and CD24, and with aldehyde dehydrogenase substrate to simultaneously detect ALDH activity.

▶ 05:44
6
Isolate cancer stem cell population by FACS

Sort viable cells using flow cytometry, selecting the ALDH+/CD44+/CD24− (or variant) population as cancer stem cells for downstream functional studies.

▶ 07:43
7
Summarize CSC isolation workflow and applications

Review the complete protocol workflow and discuss downstream applications of isolated cancer stem cells in functional assays and molecular characterization.

▶ 10:15
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