Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

Abstract

BackgroundCancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation.Methodology/FindingsTreatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear β-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF, bFGF, IL-6, IL-8, HGF, PDGF-BB, G-CSF, and SCGF-β). CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors.Conclusions/SignificanceThese findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy.

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer

“…15 Drug resistance has been previously demonstrated to be a feature of LCSC. 4,6,16 As expected, LCSCs used for this study were highly resistant to commonly used antineoplastic agents (Figure 1f), whereas their differentiated progeny and the commercial cell line H460 were overall more sensitive to drug-induced death. Altogether, these observations substantiate the validity of LCSC as cellular models in the search of new options for lung cancer therapy.…”

“…3 Lung cancer stem cells (LCSCs) have been previously identified through different criteria including surface expression of CD133, c-kit or through functional properties such as selective drug survival, elevated aldehyde dehydrogenase (ALDH) activity, increased glycolysis and glycine/serine metabolism or low concentrations of reactive oxygen species and ATP. [4][5][6][7][8][9] Importantly, when inoculated into immunocompromised mice, LCSCs give rise to xenografts that histologically reproduce the tumor of origin, thus representing an improved model for in vivo testing of new targeted therapies. 10 Several tumors express elevated levels of anti-apoptotic Bcl-2 family proteins such as Bcl-2, Bcl-X L and Mcl-1, which affect the apoptotic threshold of neoplastic cells contributing to chemotherapy resistance.…”

Oct-4+/Tenascin C+ neuroblastoma cells serve as progenitors of tumor-derived endothelial cells

“…Previous studies [20,22] have demonstrated that sidepopulation cells of the SK-NB-BE(2) NB cell line and tumor-initiating human NB cells express the embryonic stem cell [12][13][14] and cancer stem cell marker Oct-4 [15][16][17][18][19][20]. The human Oct-4 gene can generate at least three transcripts (Oct-4A, Oct-44B and Oct-4B1) and four protein isoforms (OCT4A, OCT4B-190, OCT4B-265 and OCT4B-164) by alternative splicing and alternative translation initiation [31].…”

“…Most Oct-4 + and Oct-4 − cells expressed VEGF-R2 (Supplementary information, Figure S1, [13][14][15] [8], and the neuroblastic markers GD2 (Supplementary information, Figure S1, 9-11), CD56 (Supplementary information, Figure S1, [17][18][19] [8,49,50] and NB84 ( Figure 2A). All isotypic controls used for immunofluorescence tested negative ( Figure 2B Figure S1, 4,8,12,16,20).…”

“…The Octamer-binding transcription factor 4 (Oct-4), together with SOX-2 and NANOG, maintains self-renewal and blocks cell differentiation in embryonic stem cells [12][13][14] and is expressed by some cancer stem cells [15][16][17][18][19][20]. Among these factors, Oct-4 and SOX-2 appear to be indispensable for stemness maintenance [21].…”