Reporter gene systems for the identification and characterization of cancer stem cells

Table 1 Studies using reporter system based on known cancer stem cells biomarkers

Promoter gene/element response	Reporter gene	Tumor type	Functional assays performed for validation
ALDH	Far red fluorescent protein (mNeptune)	Breast cancer[31]	Sphere formation
			Limiting-dilution xenotransplantation
			Reprogramming of non-CSCs to CSCs after cytotoxic treatments
			Extravasation potential
			Drug sensitivity in vivo
	tdTomato fluorescent protein	Breast and colon cancer[35]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vivo
	DsRed2 fluorescent protein	Oral squamous cell carcinoma[29]	Sphere formation
			Xenotransplantation assays
			Drug sensitivity in vitro
CD133	Luciferase/RFP	Glioma[16]	Sphere formation
			Transactivation assay
			Limiting-dilution xenotransplantation
			Drug sensitivity in vivo
			Drug sensitivity in vitro
AFP	GFP	Liver (cholangiocarcinoma) [94]	Sphere formationLimiting-dilution
			xenotransplantation
			Drug sensitivity in vitro
		Pancreatic cancer[95]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
Notch	GFP	Breast cancer[51]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vivo
	Luminescent protein	Lung cancer[52]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vivo
			Drug sensitivity in vitro
TERT	GFP	Osteosarcoma[53]	Sphere formation
			Limiting-dilution xenotransplantation
			Extravasation potential
			Drug sensitivity in vitro
s-SHIP	GFP	Prostate cancer[96]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
DACH1	GFP	Breast cancer[97,98]	Sphere formation
			Xenotransplantation assay
			Transactivation assay
LGR5	GFP	Colorectal cancer[99]	Sphere formation
			Gene expression profiling of flow-sorted cells

ALDH: Aldehyde dehydrogenase; AFP: Alpha-fetoprotein; TERT: Telomerase reverse transcriptase; s-SHIP: Stem-SH2-domain-containing 5'-inositol phosphatase; LGR5: G-protein-coupled receptor 49, Gpr49; GFP: Green fluorescent protein; RFP: Red fluorescent protein.

Table 2 Studies using reporter system based on CSCs transcription factors.

Promoter gene/element response	Reporter gene	Tumor type	Functional assays performed for validation
NANOG	GFP; Luminescent protein	Breast cancer[100]	Sphere formation
			Limiting-dilution xenotransplantation
			Extravasation potential
			In vitro limiting-dilution assays
		Breast cancer[98]	Transactivation assay
		Prostate cancer[70]	Sphere formation
			Extravasation potential
			Drug sensitivity in vitro
			Limiting-dilution xenotransplantation
		Nasopharynx cancer [101]	Sphere formation
			Limiting-dilution xenotransplantation
			Extravasation potential
			Drug sensitivity in vivo
			Drug sensitivity in vitro
		Liver (hepatocellular carcinoma)[45]	Sphere formationLimiting-dilution xenotransplantation
			Extravasation potential
			Drug sensitivity in vitro
		Ovary cancer[89]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
		Ovary cancer[72]	Sphere formation
			Xenotransplantation assay
			Drug sensitivity in vitro
			Extravasation potential
SOX2	tdTomato fluorescent protein/ Luminescent protein; GFP; Luminescent protein	Breast cancer[17]	Sphere formation
		Breast cancer[102]	Sphere formation
		Breast cancer[43]	Sphere formation
			Drug sensitivity in vitro
			Transactivation assay
		Breast cancer[98]	Transactivation assay
		Glioma[46]	Sphere formation
			Xenotransplantation assay
			In vitro limiting-dilution assays
		Skin cancer[103]	Limiting-dilution xenotransplantation
			Drug sensitivity in vivo
		Cervical cancer[104]	Sphere formation
			Limiting-dilution xenotransplantation
		Cervical cancer[105]	Sphere formation
			Transactivation assay
		Thyroid cancer[106]	Transactivation assay
		Teratomas from neoplastic hPSCs[12]	Transactivation assay
			Drug sensitivity in vivo
			Progenitor assays (clonogenic and multilineage hematopoietic differentiation)
OCT4	GFP; Luminescent protein	Liver (hepatocellular carcinoma)[47]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
		Melanoma[92]	Sphere formation
			Drug sensitivity in vitro
			Xenotransplantation assay
		Sarcoma[18]	Xenotransplantation assay
			Drug sensitivity in vitro
			Drug sensitivity in vivo
		Breast cancer[43]	Sphere formation
			Drug sensitivity in vitro
			Transactivation assay
		Teratomas from neoplastic hPSCs[12]	Transactivation assay
			Drug sensitivity in vitro
			Progenitor assays (clonogenic and multilineage hematopoietic differentiation)
SOX2-OCT4	GFP; mCherry fluorescent protein; Luminescent protein; Luminescent protein/ RFP	Breast cancer[38]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
		Sarcoma[42]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
		Prostate cancer[107]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vivo
			Drug sensitivity in vitro
		Gastric cancer[44]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
		Malignant mesothelioma[108]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
		Head and neck squamous cancer[109]	Sphere formation
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
		Glioma[16]	Sphere formation
			Transactivation assay
			Limiting-dilution xenotransplantation
			Drug sensitivity in vitro
			Drug sensitivity in vivo

hPSCs: Human pluripotent stem cells; GFP: Green fluorescent protein; RFP: Red fluorescent protein