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Microbiology JoVE (Open Access) Citable · DOI

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

DOI: 10.3791/57011-v
What you'll learn
  • ✓ Isolate tumor-initiating cells from sarcoma PDX using HLA-1 negative selection by FACS
  • ✓ Validate and characterize HLA-1-negative tumor-initiating cells in vitro
  • ✓ Process sarcoma tissue and establish patient-derived xenograft models
Protocol

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

Difficulty
advanced
Total time
~4-6 weeks (tissue processing and PDX establishment); ~2-3 hours per FACS isolation and sorting session
Model organism
Human sarcoma patient-derived xenografts (PDX) in immunocompromised mice
Biosafety
BSL-2

Steps

1
Process sarcoma tissue and establish patient-derived xenografts

Prepare human sarcoma tissue samples and establish PDX models in mice. This foundational step generates the tumor tissue required for subsequent cell isolation.

▶ 00:44
2
Sort and isolate HLA-1-negative tumor-initiating cells

Use fluorescence-activated cell sorting with HLA-1 as a negative marker to isolate tumor-initiating cells from PDX tissue, followed by culture in conditions that promote tumorsphere formation.

▶ 02:32
3
Characterize HLA-1-negative tumor-initiating cells

Validate and phenotypically characterize the isolated HLA-1-negative cell population through representative analyses demonstrating tumor-initiating properties.

▶ 05:11
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