Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

Background/Aims: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy

Caratterizzazione di cellule staminali tumorali ottenute da linee cellulari di carcinoma della laringe e di carcinoma della vescica

Secondo la teoria delle cellule staminali tumorali, lo sviluppo del tumore sarebbe sostenuto dalla presenza di una distinta sottopopolazione di cellule tumorali, denominate cancer stem cells (CSCs), dotate della capacità di autorigenerarsi e di una spiccata resistenza agli agenti chemioterapici. Nel presente studio, è stato messo a punto un protocollo di crescita volto all’arricchimento in CSCs, mediante formazione di sfere, delle linee cellulari Hep-2 (carcinoma della laringe), T24 (carcinoma della vescica), MG63 (osteosarcoma), CaCo-2 (carcinoma del colon-retto) e A549 (carcinoma polmonare). Successivamente è stata effettuata un caratterizzazione molecolare e fenotipica delle popolazioni arricchite in CSCs, mediante rispettivamente l’analisi di espressione di markers di staminalità e la valutazione in vivo del potenziale tumorigenico. Inoltre, a carico delle CSCs e delle cellule di controllo, sono stati analizzati i livelli dell’enzima nicotinamide N-metiltrasferasi (NNMT). L’analisi immunocitochimica e mediante Real-Time PCR hanno evidenziato elevati livelli di espressione dei markers di staminalità nelle popolazioni cellulari arricchite in CSCs rispetto ai controlli. In seguito all’inoculo in topi atimici delle cellule relative alla linea Hep-2, la popolazione arricchita in CSCs dava luogo a masse tumorali di dimensioni maggiori rispetto a quelle originatesi dalle cellule di controllo, evidenza che suggerisce la spiccata capacità da parte delle CSCs di indurre la formazione in vivo di tumori. Le analisi condotte a carico dell’NNMT mostrano un’overespressione dell’enzima nelle popolazioni arricchite in CSCs rispetto ai controlli. In considerazione dell’importante ruolo svolto dalle CSCs nello sviluppo di recidive e nella diffusione metastatica, i risultati riportati in questo lavoro potrebbero contribuire allo sviluppo di nuove strategie terapeutiche per il trattamento del cancro e suggeriscono il significativo coinvolgimento dell’NNMT nel metabolismo della cellula neoplastica.According to cancer stem cells theory, tumor development would be maintained by a distinct subpopulation of tumor cells, termed cancer stem cells (CSCs), that have the ability to self-renew and to resist to chemotherapeutic agents. In the present study, we setup a culture system to enrich CSCs from Hep-2 (laryngeal cancer), T24 (bladder cancer), MG63 (osteosarcoma), CaCo-2 (colorectal cancer) and A549 (lung cancer) cancer cell lines, through sphere formation. We further performed molecular and phenotypic characterization of CSC-enriched cell populations, by exploring the expression levels of stem markers and in vivo evaluating their tumorigenic potential, respectively. Moreover, we investigated the expression levels of the enzyme nicotinamide N-methyltransferase (NNMT) in CSCs and parental cells. Real-Time PCR and immunocytochemistry revealed that CSC-enriched populations showed increased expression levels of stem markers compared with controls. After subcutaneous injection of Hep-2 cells into immunocompromised mice, CSC-enriched population yielded tumors of a much larger size compared with those generated by parental cells, suggesting the strong ability of CSCs to form tumors in vivo. NNMT expression analysis revealed enzyme upregulation in CSC-enriched populations compared to parental counterpart. Considering the fundamental role of CSCs in tumor relapse and onset of metastases, findings reported in this work could contribute to the development of new therapeutic strategies for cancer treatment and suggest an important involvement of NNMT in cancer cell metabolism

NNMT enzyme assay.

<p>NNMT specific activity in 7 human cancer cell lines was tested by measuring the amount of N¹-methylnicotinamide produced.</p

Hematoxilyn and eosin stain of xenograft tumors generated by injection of mock (A) and transfected (B) PE/CA-PJ15 cells.

<p>(<b>A</b>) Histological examination of tumor revealed nests of cohesive neoplastic epithelial cells, with connective tissue and a band of inflammatory infiltrate. (<b>B</b>) Histological examination of tumor revealed nests of neoplastic epithelial cells with ialine connective tissue and focal inflammatory infiltrate. Original magnification ×20.</p

Effects of NNMT knockdown on the cell proliferation and colony formation <i>in vitro</i>.

<p>Cell proliferation was assessed by MTT analysis (A) and by soft-agar colony-forming assay (B). Cell growth and colony formation were evaluated in mock and NNMT silenced cells after 24–48–72 hours and 30 days of incubation, respectively. Three of the four plasmids were able to significantly reduce cell proliferation (*, p<0.05; **, p<0.005).</p

<i>In vivo</i> tumor formation of mock and silenced cells.

<p>Representative photo showing BALB/c nude mice subcutaneously injected, in the right and left flank, with mock (A) and transfected PE/CA-PJ15 cells (B), respectively.</p

Detection of NNMT knockdown.

<p>PE/CA-PJ15 cells were treated with four shRNA plasmids against NNMT (pLKO. 1-330, 1-448, 1-164, 1-711), or with transfection reagent only (mock). (A) Real-Time PCR was used to analyze the amount of NNMT mRNA in transfected compared to mock cells. (B) Protein lysates, obtained from NNMT silenced and mock cells, were analyzed for NNMT expression by Western blot.</p