Cell Biology JoVE (Open Access) Citable · DOI

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Name: In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells
Uploaded: 2026-06-17
Duration: 11 min 37 s
Description: Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

DOI: 10.3791/52446-v

What you'll learn

✓ Culture ovarian cancer cells under low-attachment, serum-free conditions to enrich tumor-initiating cells
✓ Perform flow cytometry to identify and confirm TIC marker expression in enriched populations
✓ Validate tumorigenicity of cultured TICs through subcutaneous xenograft injection in vivo

Protocol

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Difficulty

advanced

Total time

~7–14 days (including multicellular spheroid generation, marker confirmation, and in vivo validation)

Model organism

Ovarian cancer cell lines (human); immunocompromised mice for xenograft validation

Biosafety

BSL-2

Steps

Generate multicellular spheroids from ovarian cancer cell lines

Culture ovarian cancer cells in low-attachment, serum-free conditions to promote formation of three-dimensional multicellular spheroid structures enriched for tumor-initiating cells.

▶ 01:43

Stain TIC-enriched cells for flow cytometric analysis

Prepare spheroid-derived cells with fluorescently labeled antibodies against established tumor-initiating cell markers and perform flow cytometry to quantify marker expression.

▶ 05:02

Confirm tumorigenicity via subcutaneous xenograft injection

Inject cultured TIC populations subcutaneously into immunocompromised mice and monitor for tumor formation to validate in vivo tumorigenic potential of the enriched cell population.

▶ 07:39

Analyze enrichment and tumorigenicity results

Evaluate flow cytometry data and in vivo tumor take rates to confirm that low-attachment, serum-free culture conditions successfully enrich for cells with TIC markers and functional tumorigenicity.

▶ 09:03

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