Could gastrointestinal tumor-initiating cells originate from cell-cell fusion in vivo?

Table 1 Tumor initiating cell origination from cell-cell fusion

Cell I	Cell II	Cell fusion method	Cell fusion evidence	Tumor initiating method	Tumor initiating evidence	Mechanism	Ref.
Colorectal cancer cell	Human dendritic cell	In vitro. DCs and cancer cells fused using PEG	Purified cells		Enhanced induction of antigen-specific CTL	Streptococcal preparation OK-432 promotes fusion efficiency	[69]
Human esophageal carcinomas cell	Human dendritic cell	In vitro. ECs and DCs fused using PEG	Co-expression of MHC class II, CD86, and MUC1		Induced specific antitumor response		[55]
Human gastrointestinal cancer cell	Human dendritic cell	In vitro. Fusion via PEG and electroporation			Th1/Th2 and Tc1/Tc2 balance improved		[73]
Human gastric cancer cell	Human dendritic cell		FACS analysis		Induced CTLs, reduced metastases		[56]
Human gastric cancer cell (HGC-27 or SGC-7901)	hucMSC	In vitro. GC-DIO and hucMSCs-DID fusion using PEG	Double positive cells	BALB/C nude mice (2 × 106 cells)	In vivo. Tumors formed from fused cells		[44]
Human breast cancer cell (MDA-MB-231)	Human MSC	In vivo. 2 × 106 MSC300415-GFP and 2 × 106 MDA-MB-231-cherry subcutaneously injected into 5 female NOD/SCID mice	Hybrid cells GFP/cherry fluorescence	1 × 106 MDA-hyb3-GFP/cherry cells were injected subcutaneously into 3 female NOD/SCID mice	In vivo. Tumors formed from fused cells		[80]
Human colon adenocarcinoma cell	Human HeLa cell (D98OR)	In vitro. Fused using PEG, isolation of hybrid cells by selecting isolated colonies	Flow cytometry analysis had more DNA than expected. A range of 71–140 chromosomes		Fusion cell characteristics were consistent with cancer cells		[75]
Human colon cancer cell (SW480)	Human dendritic cell	In vitro. Tumor cells- PKH26-red and DCs- PKH67-green fused using PEG	Dual red and green fluorescence and highly expressed CD80, CD86, and HLA-DR	CD8+ T lymphocytes co-cultured with dendritic cells at a ratio of 10:1	CTLs were activated to proliferate and the number of T cells increased		[55]
Human colon cancer cell (SW620)	Human dendritic cell	In vivo. DCs and tumor cells fused using PEG	Fusion efficacy was evaluated by FM and FC	In vivo. 1 × 107 fusion hybrids injected intraperitoneally	Cellular immune responses, significant inhibition of tumor growth		[55]
Human colon carcinoma line (VACO-411)	Human breast cancer line (MCF-7)	In vitro. VACO-411 (1 × 106 cells) and MCF-7 (1 × 106 cells) fused using PEG	Morphology of VACO-411 × MCF-7 fused cells	In vitro. The fused cells were treated with TGF-β	Fusion cells were inhibited by TGF-β		[76]
Human colon epithelial cancer cell	Human normal colon cell	In vitro. Cancer cells and normal cells (1:10) fused using PEG	Comparison of DNA synthesis (P < 0.01)	Male mice nu/nu injected subcutaneously with1 × 106 fused cells	The fused cells could not grow into tumors		[77]
Human colorectal carcinoma cell	Human dendriticcell	In vitro. DCs-CMFDA-green,colorectal carcinoma-CMTMR-red cells fused using PEG/electrofusion	Double-positivecells		Efficiently activated autologous tumor-specific T cells		[68]
Human esophageal cancer cell (EC109)	Human dendritic cell	In vitro. DCs and ECs (5:1-10:1) fused using PEG	Co-expression of MHC-CiaSSII and CD86 and MUC1 antigens	Cytotoxic T lymphocytes	Antitumor capabilities		[60]
Human esophageal cancer cell (EC9706)	hucMSC	In vitro. ECs-DiO hMSCs- DiD fused using PEG	Double positive hybrids are yellow and multinuclear	In vivo. Xenograft assays in immunodeficient mice	Both ECs and their self-fusion groups developed tumors
Human esophageal carcinoma cell (EC9706)	Human hemopoietic stem cell	In vitro. ECs and HSCs (10:1) fused using PEG	CD34+CD38-Scal+ cells isolated using immunomagnetic beads; HSCs cannot grow in DMEM supplemented with 10% FBS	In vivo. 5 × 105 fused cells injected into 12 NOD/SCID mice	All mice formed tumors; however, the tumor weight of the fused cell group was lower than that of the EC9706 group		[54]
Human esophageal carcinoma cell	Human dendritic cell	In vitro. DCs and ECs (5:1) fused using PEG; incubated with FA-FITC and CD80-PE	Analysis using FATICan	In vivo. Fusion vaccine (0.2 mL; 1 × 106 cells) injected	Anti-tumor effects		[58]
Human esophageal carcinoma cell (EC109)	Human dendritic cell	In vitro. DCs and ECs (5:1) fused using PEG. FA-FITC CD80-PE mAbs-CD80, CD83 and CD86	FC	In vitro. Cytotoxicity assays	Antitumor activity		[59]
Human female pancreatic adenoepithelial neoplasm cell	Human male BMDC	In vivo. 4 female pancreatic cancer patients transplanted with male BMDCs	Peripheral blood cell: EpCAM (yellow)/CD45 (green), Y chromosome, CK+/CD45+, MФ proteinsCD14, CD16, CD11c, CD163 MUC4				[43]
Mouse colon cancer cell (MC38)	Mouse R26R- YFP Cre mice	In vivo. MC38 cells were injected ventrally into r26R-YFP Cre mice	RFP+ YFP+				[51]
Mouse primary melanoma cell	Mouse MФ	Melanoma cells were injected into mice intradermally	RFP and GFP cells	300 and 3000 cells, respectively injected into mice (n = 9, 3)	Tumor initiation	The characteristics of parental cells provided adhesive affinity	[51]
Human gastric cancer cell (MKN-1)	Dendritic cell	In vitro. DCs- PKH-26 and GC cells-PKH-67 fused via PEG/electrofusion	Double positive cell populations	In vitro. Co-cultured DCs (1 × 105 cells) with 1 × 106 T cells	Induced tumor antigen-specific CD8+ T cells		[70]
Human gastric epithelial cell (GES-1)	CM-MSC	In vitro. GES-1- PHK-26 (2 × 106 cells) and CM-MSCs- CFSE (1 × 107) cells fused using PEG	Most cells express PKH26 and CFSE	In vivo. The fused cells (1 × 107cells) were injected into 8 BALB/c nude mice	Tumors from the fused cells formed in vivo		[47]
Human gastric cancer cell (SGC7901)	Human dendritic cell	In vitro. SGC7901 and DCs fused using PEG	Pure fused cells were obtained by selective culture with HAT/HT culture system	In vivo. Fusion cells (5 × 108) were injected into BALB/c mice	In vivo. The fused cells could not grow into tumors		[61]
Human gastric cancer cell (SGC7901)	Human dendritic cell	In vitro. SGC7901 and DCs fused using PEG	Selective culture with the HAT/HT culture system	In vivo. Fused cells (5 × 106/mL) + T cells (5 × 106/mL)	In vivo. The fused cells could not grow into tumors		[62]
Human hepatobiliary stem/progenitor cell	Human hematopoietic precursor-derived myeloid intermediate	In vitro. Cultures of CD34+ LTICs and xenograft cells (the xenografts were produced by CD34+ hybrid cells)	CD34+ LTICs co-expressed liver stem cell and myelomonocytic cell markers			HSPCs were fused with a CD34+ hematopoietic precursor-derived myeloid intermediate to form CD34+ hybrid cells
Human hepatocellular carcinoma cell (HepG2)	Human embryonic stem cell	In vitro. HepG2-red mitochondrion selective probe and hESCs-Oct-GFP cells fused via laser-induced single-cell fusion	Transfer of cytoplasmic GFP from hESCs to HepG2 cells	In vivo. HepG2 cells and the fused cells (5 × 104 and 1 × 105 cells, respectively) were injected into nude mice	Tumors were generated from fused cells
Human hepatocellular carcinoma cell (HepG2)	Mouse MSC	In vitro. MSCs- DiI (5 × 105 cells) and HepG2-eGFP (1 × 105 cells) fused using PEG	Dual fluorescence, two nuclei	In vivo. The fused cells were injected into 7 nude mice/group with 2.4 × 107 cells/group	Tumors were formed from fused cells
Human intestinal cancer cell (HT-29)	Human MSC	In vitro. PM7-eGFP and HT-29-DsRED cells were cocultured	eGFP and DsRED double positive cells		Acquired epithelial characteristics		[51]
Human intestinal epithelial cells	Human hematopoietic cell	In vitro. X- and Y-chromosome determined by FISH. Female recipients of hematopoietic cell transplant from male donors	Stained for X- (green) and Y- (red) chromosomes and Lamin B1 (white)			In mice, hematopoietic fusion with non-hematopoietic cell types occurs endogenously in the absence of disease	[86]
Mouse intestinal epithelial cell	Mouse bone marrow-derived cell	In vivo. CMV-CreGFP+ mice BM were transplanted into iDTR mice	Co-staining for GFP and EpCAM.GFP+ cells in the intestine		Cell fusion is dispensable for tissue homeostasis		[52]
Mouse intestinal stem cell	Human bone marrow-derived cell	In vivo. Donor female mice BMDCs-GFP, male recipient mice	EGFP expression in all principal intestinal epithelial lineages				[63]
Mouse colon adenocarcinoma cell (CT26)	Mouse dendritic cell	In vitro. Tumor cells- PKH67-Green and DCs fused using PEG	Assessedvia the trypan-blue exclusion test	In vivo. BALB/c mice injected with 5 × 105 cells	The fused cells could not generate tumors		[64]
Mouse colon adenocarcinoma cell line (CT26)	Mouse dendritic cell	In vitro. DCs-anti-CD11cmAb and tumor cells- CFSE fused using PEG	Analyzed by FC	In vivo. Injection of 1 × 104, 105, or 106 cells/mouse	The fused cells did not generate tumors. CTL anti-tumor effects		[72]
Mouse colon carcinoma cells (CT26CL25)	Allogeneic and semi allogeneic dendritic cells	In vitro. DCs-PKH26-red andCT26CL25-PKH67-green fused using PEG	Analyzedby FC	In vivo. 1 × 106 fused cells and 5.0 × 105 CT26CL25 cells	Anti-tumor effects in vivo		[65]
Mouse colon epithelial cell	Mouse BMDC	In vivo. Female mice BMDCs-GFP (1 × 107 cells) transplanted into irradiated male mice	Co-expression of GFP and the Y chromosome	In vivo. Parabiosis surgery (GFP and ROSA mice)	Bone-marrow/epithelial cell fusion causes genetic reprogramming	Inflammation and proliferation act together to mediate intestinal cell fusion	[87]
Mouse gastric epithelial cell	Mouse BMDC	In vivo. Male irradiated C57BL/6 mice received female C57BL/6 mice BMDC-GFP	Direct. Positive for the Y chromosome and expressed GFP as determined by FM	In vivo. GCs were induced with a carcinogen	Tumor formed from fused cells in vivo	Chronic inflammation (adenocarcinoma, glandular stomach, not squamous cell carcinoma)	[48]
Mouse hepatocellular carcinoma cell	Mouse dendritic cell	In vitro. HCCs PKH-26-red and DCs-PKH-2-green fused using PEG	The fusion cells were yellow under the confocal microscope	In vitro. CTL assay	In vitro. Activated cytotoxic T lymphocytes		[66]
Mouse hepatoma cell line (Hepa1-6)	Mouse embryonic stem cell	In vitro. Cancer cells-GFP and ES cells-RFP fused using PEG	Double fluorescence-positive	In vivo. 1 × 106 ES-cancer fused cells injected into nude mice	Tumor formed from fused cells in vivo
Mouse intestinal epithelial cancer cell	Mouse WBM (macrophage)	In vivo. WBM-GFP (5 × 106 cells) injected in recipient mice (male WT, ApcMin/+, ROSA26, ROSA26/ApcMin/+). Parabiosis	Co-localization of GFP (green) and β-galactosidase (red)		Nuclear reprogramming	Fusion between circulating blood-derived cells and tumor epithelium origin at the natural course of tumorigenesis	[39]
Mouse intestinal epithelial cells (IEC-6). Human cervical adenocarcinoma cells (HeLa)	Mouse intestinal epithelial cells (IEC-6) Human cervical adenocarcinoma cells (HeLa)	In vitro. IEC-6- CFSE andIEC-6- SNARF-1 (HeLa -Cy3-dUTP-red and HeLa- Cy5-dUTP-green) fused using PEG	The fused cell emits both CFSE and SNARF-1 fluorescence (IEC-6). Eight daughter cells contain both dyes (HeLa)	In vivo. The IEC-6 fused cells (Two million cells) were injected in 18 mice	Tumor formed from the fused cells in vivo (n = 11 generated tumors)		[78]
Mouse intestine stromal cell	Mouse bone marrow-derived macrophage	In vivo. Female mice BMDCs-GFP injected in male recipient mice	Co-localization of GFP and Y-chromosome		Organ fibrosis	Depleting macrophages genetically reduced the number of cells	[53]
Mouse prostate cancer cell (PCa)	Mouse BMDC	In vivo. 2 × 106 cells/mice BMDCs-GFP transplanted into 10 C57BL/6 mice	Co-expression of GFP and CK8	C57BL/6 mice-GFP, induced prostate cancer by MNU	GFP-positive cells in the prostate cancer tissue		[79]
Whole tumor cell	Human dendritic cell	In vitro. The purified DCs and tumor cells fused using PEG					[67]

BMDC: Bone marrow-derived cell; CAM: Cell adhesion molecules; CM-MSCs: Cord matrix-derived mesenchymal stem cells; CTL: Cytotoxic lymphocytes; DC: Dendritic cell; DMEM: Dulbecco's modified eagle medium; EC: Esophageal carcinoma; FACS: Fluorescence-activated cell sorting; FBS: Fetal bovine serum; FM: Fluorescence microscope; FC: Flow cytometry; GC: Gastric cancer; hucMSCs: Human umbilical cord mesenchymal stem cells; HSPC: Hematopoietic stem and progenitor cell; hESC: Human embryonic stem cell; HSC: Hematopoietic stem cell; HCC: Hepatocellular carcinoma; HLA: Human leukocyte antigen; LTICs: Liver tumor-initiating cells; MSC: Mesenchymal stem cell; MNU: N-methyl-N-nitrosourea; PEG: Polyethylene glycol; WBM: Whole bone marrow; WT: Wild-type; IEC: Intestinal epithelial cell; HGC: Human gastric cancer cell; GFP: Green fluorescent protein; MDA: Malonaldehyde; TGF: Transforming growth factor; FITC: Fluorescein isothiocyanate; PE: Physical examination; CM: Chylomicron.

Table 2 Molecules potentially involved in gastrointestinal tumor-initiating cell generation from cell-cell fusion

Tumor type	Cell surface molecules involved in cell fusion	Intracellular molecules involved in cell fusion	Signaling pathways involved in cell fusion	giTIC molecules
Gastric	CD44, CD133, EpCAM, CXCR4, Lgr5, CD54[3], ALDH1[3]	OCT4, SOX2[120], Twist, Slug[47], Nanog, Lin28[44]	CXCL12/CXCR4, Lgr5[3]	CD44[3], CD133[111]
Colorectal	CD44, CD133, EpCAM, CXCR4, CD166[3]. CD81, CD9, GTP-binding protein α13, radixin[85], Syncytin 1, CD47	APC, p53, Kras, NF-κB, OCT4, SOX2[3]. ADAM10, myosin regulatory light chain, RhoA[85]	CXCL12/CXCR4, Wnt/β-catenin[3], c-Jun	CD133, CD44, ALDH1, EpCAM, CD44, CD166, CD24, LGR5, CD26[3]
Liver	CD44, CD133, CD13, EpCAM, CD24, CD90[3], E-cadherin, matrix metalloproteinase	p53[3], OCT4, SOX2[119]. Vimentin, Twist, Snail[113]	CXCL12/CXCR4[77], Wnt, TGF-β, Notch, Hedgehog[3]	CD133, CD49f, CD90, CD13[3]
Esophageal	CD44, CD133, EpCAM[115], CXCR4[3]	OCT4, SOX2	Lgr5[3], CXCL12/CXCR4	CD44, ALDH1[3]
Pancreatic	CD44, CD133, EpCAM, CXCR4, CD24[3], ALDH1[3]	KRAS, TP53, SMAD4, OCT4, SOX2[116]	CXCL12/CXCR4[3], Lgr5[118]	CD133, CD44, CD24, ESA, CXCR4[3]

giTIC: Gastrointestinal tumor-initiating cell; CD44 and CD133: Cell surface glycoproteins; CXCL12: Chemokine; CXCR4: Chemokine receptors 4; EpCAM: Epithelial cell adhesion molecule, transmembrane glycoprotein; ALDH: Acetaldehyde dehydrogenase; NF-κB: Nuclear factor kappa-B; APC: Adenomatous polyposis coli; GTP: Guanosine triphosphate; ESA: Epithelial-specific antigen; TGF-β: Transforming growth factor-β.