Lung Cancer Stem Cells Research Articles

NEAT1 acts as an inducer of cancer stem cell-like phenotypes in NSCLC by inhibiting EGCG-upregulated CTR1.

Long non-coding RNAs (lncRNAs) play significant roles in the pathogenesis of various cancers, including lung cancer. In this study, we aimed to investigate the biological function of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in cancer stem cells (CSCs). CSCs have been suggested as the main cause of tumor metastasis, tumor recurrence, and chemotherapy resistance. The copper transporter 1 (CTR1) has been the focus of many recent studies because of its correlation with cisplatin (CDDP) resistance. So far, the mechanism of how NEAT1 regulates CSCs in NSCLC remains unknown. In the current study, lung cancer stem cells were enriched from the parental NSCLC cells. We observed that NEAT1 was up-regulated while copper transporter 1 (CTR1) was down-regulated in the enriched NSCLC cancer stem cells. Knockdown of NEAT1 was able to decrease the CSC-like properties in NSCLC cells, while over-expression of NEAT1 could contribute to the stemness respectively. Meanwhile, appropriate doses of EGCG restrained the stemness triggered by over-expressing NEAT1 via inducing CTR1 expression. Wnt signal pathway and epithelial-to-mesenchymal transition (EMT) process were involved in NEAT1-induced CSCs in NSCLC. These findings may suggest a novel role of NEAT1 for NSCLC treatment.

Upregulation of FoxM1 by MnSOD Overexpression Contributes to Cancer Stem-Like Cell Characteristics in the Lung Cancer H460 Cell Line.

Manganese superoxide dismutase promotes migration and invasion in lung cancer cells via upregulation of the transcription factor forkhead box M1. Here, we assessed whether upregulation of forkhead box M1 by manganese superoxide dismutase overexpression mediates the acquisition of cancer stem-like cell characteristics in non-small cell lung cancer H460 cells. The second-generation spheroids from H460 cells were used as lung cancer stem-like cells. The levels of manganese superoxide dismutase, forkhead box M1, stemness markers (CD133, CD44, and ALDH1), and transcription factors (Bmi1, Nanog, and Sox2) were analyzed by Western blot. Sphere formation in vitro and carcinogenicity of lung cancer stem-like cells were evaluated by spheroid formation assay and limited dilution xenograft assays. Knockdown or overexpression of manganese superoxide dismutase or/and forkhead box M1 by transduction with short hairpin RNA(shRNA) or complementary DNA were performed for mechanistic studies. We showed that manganese superoxide dismutase and forkhead box M1 amounts as well as the expression levels of stemness markers and transcription factors sphere formation in vitro, and carcinogenicity of lung cancer stem-like cells were higher than in monolayer cells. Lung cancer stem-like cells transduced with manganese superoxide dismutase shRNA or FoxM1 shRNA exhibited decreased sphere formation and lower amounts of stemness markers and transcription factors. Overexpression of manganese superoxide dismutase or FoxM1 in H460 cells resulted in elevated sphere formation rates and protein levels of stemness markers and transcription factors. Meanwhile, manganese superoxide dismutase knockdown or overexpression accordingly altered forkhead box M1 levels. However, forkhead box M1 knockdown or overexpression had no effect on manganese superoxide dismutase levels but inhibited or promoted lung cancer stem-like cell functions. Interestingly, forkhead box M1 overexpression alleviated the inhibitory effects of manganese superoxide dismutase knockdown in lung cancer stem-like cells. In a panel of non-small cell lung cancer cells, including H441, H1299, and H358 cells, compared to the respective monolayer counterparts, the expression levels of manganese superoxide dismutase and forkhead box M1 were elevated in the corresponding spheroids. These findings revealed the role of forkhead box M1 upregulation by manganese superoxide dismutase overexpression in maintaining lung cancer stem-like cell properties. Therefore, inhibition of forkhead box M1 upregulation by manganese superoxide dismutase overexpression may represent an effective therapeutic strategy for non-small cell lung cancer.

Zeb1 Regulates the Symmetric Division of Mouse Lewis Lung Carcinoma Stem Cells through Numb mediated by miR-31.

Symmetric cell division (SD) and asymmetric cell division (ASD) were the unique characteristics of stem cells and the mechanisms underlying stem cell renewal. While recent studies have identified the presence of SD and ASD in lung cancer stem cells (CSCs), the mechanisms regulating SD and ASD in cancer state have not been elucidated, mostly due to the lack of stable cellular models of SD and ASD in CSC research. In this study, the interaction between Zeb1, an Epithelial-Mesenchymal Transition (EMT) factor shown to regulate CSCs self-renew, and Numb, which regulates SD and ASD in the normal neural stem cell was investigated using the stable mouse Lewis lung adenocarcinoma SD (LLC-SD) and ASD (LLC-ASD) lines established from our previous study. The most significant finding derived from this line of research is that we have identified and molecularly ordered the axis of Zeb1-miR-31-Numb that regulates the SD, a mechanism of CSC self-renewal that has not been previously described. More specifically, the expression of Zeb1 and Numb were both significantly higher in LLC-SD than LLC-ASD cells. Silencing of Zeb1 or Numb expression lead to decreased ratio of SD and weakened single-cell cloning formation, tumor growth and tumor metastasis, respectively. The rescure experiments have molecularly ordered the regulation of Numb by Zeb1, indirectly mediated by miR-31. Moreover, we also provided preliminary evidence supporting the clinical relevance of our finding. In summary, our study provides a new insight for the self-renew of lung CSCs in which SD is regulated by the axis of Zeb1-miR-31-Numb.

CCAT1 stimulation of the symmetric division of NSCLC stem cells through activation of the Wnt signalling cascade.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortalities worldwide, yet this condition remains a poorly understood malignancy, and the subgroup of cancer stem cells (CSCs) leading to therapeutic resistance and adverse prognosis have not been well studied. CSCs frequently undergo symmetric division, which facilitates expansion of the stem cell pool, contributing to long-term relapse and therapy failure. CCAT1 could act as a miRNA sponge to influence downstream genes; however, its roles in NSCLC stem cell are unclear. We first identified activation of Wnt signalling in NSCLC. Analysis of the clinical data from a public database showed a significant decrease of the Wnt signalling repressor Let-7c. Using biological and informatics analyses, we hypothesized that CCAT1 stimulated the main factors of the Wnt signalling pathway, of which the three most deregulated genes were further confirmed by western blotting. Axitinib, a Wnt signalling inhibitor, effectively stimulated asymmetric division, similar to Let-7c. CCAT1 inhibition decreased the ratio of symmetric division of stem cells, and both Let-7c and Axitinib significantly abolished CCAT1 induction of symmetric division by inhibiting Wnt signalling. Restoration of Let-7c blocked the CCAT1 effects, forming the CCAT1/Let-7c/Wnt regulatory axis to control the division of lung cancer stem cells. Stimulation of stem cells to divide asymmetrically by delivering Let-7c or suppressive Axitinib could represent prospective strategies for curing lung cancer patients.

Lung cancer stem cells – the role in pathogenesis and progressive growth of cancer

Lung cancer is the main cause of cancer death worldwide. Rapid accurate diagnosis, recognition of risk factors and improvement of treatment efficacy represent the main challenges. An advanced stage of the disease at the time of diagnosis, observed in the majority of cases, makes the introduction of radical treatment impossible or ineffective. Immunotherapy was a breakthrough in achieving long-term survival in recent years. The reason for developing this type of treatment is to know the interaction between the host cells and tumor cells. Cancer stem cells theory is increasingly gaining greater significance in the world. The biological heterogeneity of lung cancer may be due to the presence of a small percentage of cancer stem cells (CSCs). CSCs are the results of genetic and epigenetic changes in normal stem cells, progenitor cells or differentiated cells. CSCs are functionally defined by their unlimited self-renewal capacity, multiple differentiation and their ability to imitate tumors. It is believed that CSCs determine unrestricted growth of tumors and their different morphology. Their ability to self-renew can be the cause of relapses even after long periods of remission. CSCs reside in niches, where they receive signals for differentiation and proliferation process. The identification of CSCs is based on the presence of specific molecules (CD133, CD44, CD90, ALDH, EpCAM). An important feature of CSCs is their resistance to conventional cancer treatment: radio therapy and chemotherapy. Presumably, CSCs are responsible for the synthesis of immunosuppressive particles, and recruiting other molecules, which have immunosuppressive properties. The paper presents the current state of knowledge on CSCs in lung cancer detailing adenocarcinoma.

Garcinol inhibits cancer stem cell-like phenotype via suppression of the Wnt/β-catenin/STAT3 axis signalling pathway in human non-small cell lung carcinomas

Innate or acquired drug resistance and consequent tumor relapse in lung cancer patients have been linked to activities of cancer stem cells (CSCs). Therefore, targeting CSCs is suggested as an effective approach for non-small cell lung cancer (NSCLC) therapy. In this study, we demonstrated that garcinol, a polyisoprenylated benzophenone isolated from fruiting bodies of Garcinia indica, and possessing anti-inflammatory, antioxidant, acetyltransferase inhibitory, and anticancer activities, modulates activities of lung CSCs (LCSCs) and their associated aggressiveness. Herein, we demonstrated the inhibitory effect of garcinol on the LCSC phenotype of human NSCLC cells using analytical drug cytotoxicity or cell viability, flow cytometric, and functional assay approaches. Garcinol significantly diminished the ability of the H441 and A549 NSCLC cell lines to form spheres. In parallel assays, garcinol inhibited differentiated lung cancer cell and LCSC viability in dose-dependent manners. Consistent with these observations, flow cytometric data showed that garcinol reduced the putative LCSC pool, evidenced by the dose-dependent decreasing proportion of side-population (SP) cells and associated ALDH activity in garcinol-treated H441 cells, compared to the control group. Additionally, functional assays showed that garcinol markedly diminished the ability of H441 and A549 cells to form colonies. Mechanistically, garcinol impaired phosphorylation of LRP6, a co-receptor of Wnt and STAT3. In the same assay, garcinol down-regulated β-catenin, Dvl2, Axin2, and cyclin D1 expressions in NSCLC-generated spheres, suggesting its ability to regulate the Wnt/β-catenin signaling pathway. The results were further verified in vivo using H441 LCSC mouse xenograft model where administration of garcinol significantly inhibited tumor growth. Taken together, we demonstrated herein that garcinol modulates the LCSC phenotype via regulation of Wnt/β-catenin signaling and inactivation of STAT3, thus showing that garcinol may be a putative novel anti-LCSC therapeutic agent.

Lung Cancer Stem Cells: An Epigenetic Perspective.

Lung cancer remains the major cause of human mortality among all the cancer types despite the colossal amount of efforts to prevent the cancer onset and to provide the appropriate cure. Recent reports have identified that important contributors of lung cancer-related mortality are the drug resistance and aggressive tumor relapse, the characteristics contributed by the presence of lung cancer stem cells (CSCs). The identification of lung CSCs is inherently complex due to the quiescent nature of lung epithelium, which makes the distinction between the normal lung epithelium and lung CSCs difficult. Recently, multiple researches have helped in the identification of lung CSCs based on the presence or absence of certain specific types of stem cell markers. Maintenance of lung CSCs is chiefly mediated through the epigenetic modifications of their genome. In this review, we will discuss about the origin of lung CSCs and the role of epigenetic modifications in their maintenance. We will also discuss in brief the major lung CSC markers and the therapeutic approaches to selectively target this population of cells.

MiR-5100 increases the cisplatin resistance of the lung cancer stem cells by inhibiting the Rab6.

Cisplatin-based chemotherapy is the most commonly used treatment regimen for lung cancer. Cancer stem cells (CSCs) are postulated to be important promoters of drug resistance. We previously found that miR-5100 is overexpressed in lung cancer, but it is unknown whether and how miR-5100 regulates cisplatin resistance. Here, we demonstrated that miR-5100 was significantly up-regulated in CD44+ CD133+ lung cancer stem cells (LCSCs) compared with non-CSCs. Additionally, over-expression of miR-5100 increased CSC properties, cell growth, and tumor sphere formation in lung cancer cell line A549 or H1299, and that miR-5100 inhibitor significantly increased sensitivity of LCSCs to cisplatin in vitro. Surprisingly, the combination with miR-5100 inhibitor significantly decreased the IC50 of LCSCs to cisplatin. Furthermore, miR-5100 increased CSC properties and cisplatin resistance by inhibiting Rab6, a direct target gene of miR-5100. We demonstrated that miR-5100 overexpression increases the cisplatin resistance of the LCSCs through the mitochondrial apoptosis pathway. In conclusion, our results suggest that miR-5100 increases the cisplatin resistance of the LCSCs by inhibiting the Rab6. This study provides novel insight into the regulation of LCSCs by miRNA.

MicroRNA-128 suppresses paclitaxel-resistant lung cancer by inhibiting MUC1-C and BMI-1 in cancer stem cells.

The existence of cancer stem cells (CSCs) is the main reason for failure of cancer treatment caused by drug resistance. Therefore, eradicating cancers by targeting CSCs remains a significant challenge. In the present study, because of the important role of BMI-1 proto-oncogene, polycomb ring finger (BMI-1) and C-terminal Mucin1 (MUC1-C) in tumor growth and maintenance of CSCs, we aimed to confirm that microRNA miR-128, as an inhibitor of BMI-1 and MUC1-C, could effectively suppress paclitaxel (PTX)-resistant lung cancer stem cells. We showed that CSCs have significantly higher expression levels of BMI-1, MUC1-C, stemness proteins, signaling factors, and higher malignancy compared with normal tumor cells. After transfection with miR-128, the BMI-1 and MUC1-C levels in CSCs were suppressed. When miR-128 was stably expressed in PTX-resistant lung cancer stem cells, the cells showed decreased proliferation, metastasis, self-renewal, migration, invasive ability, clonogenicity, and tumorigenicity in vitro and in vivo and increased apoptosis compared with miR-NC (negative control) CSCs. Furthermore, miR-128 effectively decreased the levels of β-catenin and intracellular signaling pathway-related factors in CSCs. MiR-128 also decreased the luciferase activity of MUC1 reporter constructs and reduced the levels of transmembrane MUC1-C and BMI-1. These results suggested miR-128 as an attractive therapeutic strategy for PTX-resistant lung cancer via inhibition of BMI-1 and MUC1-C.

P1.02-067 FGF1 and IGF1 Co-Stimulation Promoted the Amplification and Cancer Stemness of Lung Cancer Cells under 3D Culture Condition

Lung cancer stem cells (LCSCs) are considered as the cellular origins of metastasis and relapse of lung cancer. However, because of relative few amount of stationary LCSCs in primary cancer tissues, it is difficult to conduct large-scale mechanical and translational studies directly without any amplification of LCSCs in vitro. Routine two-dimensional culture system (2D-culture) hardly mimics the growth and functions of LCSCs in vivo and therefore significantly decreases the stemness activity of LCSCs.

The combination therapy of salinomycin and gefitinib using poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles for targeting both lung cancer stem cells and cancer cells.

PurposeLung cancer (LC) is the leading cause of cancer death worldwide. Evidences suggest that both LC cancer stem cells (CSCs) and cancer cells are supposed to be eliminated to achieve superior treatment effect against LC. Salinomycin could eradiate CSCs in various types of cancers, and gefitinib is a first-line therapy in LC. The purpose of the present study was to develop salinomycin-loaded nanoparticles (salinomycin-NPs) combined with gefitinib-loaded nanoparticles (gefitinib-NPs) to eradicate both LC CSCs and cancer cells.MethodsSalinomycin and gefitinib were encapsulated separately by poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles by the emulsion/solvent evaporation approach. The anti-LC activity of salinomycin-NPs and gefitinib-NPs was investigated.ResultsSalinomycin-NPs and gefitinib-NPs are of ~140 nm in size, high drug encapsulation efficacy and sustained release of drugs. CD133+ LC CSCs showed the characteristics of CSCs, including significantly enhanced stem cell gene expression, tumorsphere formation ability, and tumorigenicity in mice. Both salinomycin and salinomycin-NPs are capable of selectively inhibiting LC CSCs, as reflected by their enhanced cytotoxic effects toward CD133+ LC CSCs and ability to reduce tumorsphere formation in LC cell lines, whereas gefitinib and gefitinib-NPs could significantly inhibit LC cells. Salinomycin-NPs and salinomycin could reduce the population of LC CSCs in the tumors in vivo. It is noteworthy that salinomycin-NPs combined with gefitinib-NPs inhibited the growth of tumors more efficiently compared with salinomycin combined with gefitinib or single salinomycin-NPs or gefitinib-NPs.ConclusionSalinomycin-NPs combined with gefitinib-NPs represent a potential approach for LC by inhibiting both LC CSCs and cancer cells.

P2.13-001 Herbal Compound as a Potential Lead Targets Lung Cancer Stem Cells

P2.13-001 Herbal Compound as a Potential Lead Targets Lung Cancer Stem Cells

P1.02-065 Histone Deacetylase Inhibition Alters Stem Cell Phenotype in Gefitinib-Resistant Lung Cancer Cells with EGFR Mutation

Cancer stem cells (CSCs) are epigenetically altered by histone deacetylase (HDAC), resulting in chromatin condensation. We have reported that gefitinib-resistant-persisters (GRP) population within non-small cell lung cancer (NSCLC) cells possesses CSCs features. The purpose of this study is to examine the role of HDAC in the gefitinib-resistant lung CSC.

Sulforaphane reverses gefitinib tolerance in human lung cancer cells via modulation of sonic hedgehog signaling.

Gefitinib is a targeted anticancer drug that was developed as an effective clinical therapy for lung cancer. Numerous patients develop gefitinib resistance in response to treatment. Sulforaphane (SFN) is present in cruciferous vegetables, and has been demonstrated to inhibit the malignant growth of various types of cancer cells. To investigate the role of SFN in gefitinib resistance, a gefitinib-tolerant PC9 (PC9GT) cell model was established by continually exposing PC9 cells to gefitinib. Cell viability was measured using a cell proliferation assay. Components of the sonic hedgehog (SHH) signaling pathway and markers of lung cancer stem cells were detected via western blotting. SFN markedly inhibited the proliferation of PC9GT and PC9 cells in a dose-dependent manner; combination SFN/gefitinib treatment also markedly decreased PC9GT cell proliferation, compared with SFN or gefitinib administered alone (P<0.05). Western blot analysis revealed that the expression of SHH, Smoothened (SMO), zinc finger protein GLI1 (GLI1), GLI2, CD133 and CD44 were upregulated in PC9GT cells, as compared with in PC9 cells. Furthermore, SFN markedly inhibited the expression of SHH, SMO and GLI1 in PC9GT and PC9 cells in a dose dependent manner, and SFN combined with gefitinib markedly inhibited the expression of SHH, SMO, GLI1, CD133 and CD44 in PC9GT cells when compared with SFN or gefitinib monotherapy. The results of the present study demonstrated that SFN inhibits the proliferation of gefitinib-tolerant lung cancer cells via modulation of the SHH signaling pathway. Therefore, combined SFN and gefitinib therapy may be an effective approach for the treatment of lung cancer.

Lung Cancer Stem Cells: Insights into Characterization and Regulatory Mechanisms

Lung cancer is the leading cause of cancer-related mortality worldwide. The five-year survival rate is very low in lung cancer patients due to diagnosis at late stages, tumor recurrence, and drug resistance. Recent studies suggest that cancer cells with self-renewal capacity contribute to tumor growth. Identification of reliable markers for cancer stem cell populations and mechanistic understanding of associated signaling pathways and regulatory factors are critically needed to improve long-term survival. Several studies have established the expression of stem cell markers (aldehyde dehydrogenase ALDH1, glycoprotein CD44 and CD133) in lung cancer cell lines and clinical samples. Association of these stem cell markers with lung cancer and the signaling pathways (Wnt, Notch, and Hedgehog) maintaining stem cell characteristics are briefly reviewed here. Interestingly, epigenetic mechanisms and regulatory roles of transcription factors such as Sox2 and Runx family members have been shown to contribute to stem cell properties and lung cancer. Understanding mechanisms regulating “stemness” and differentiation of lung epithelial cells will lead to novel therapies for successful targeting of cancer stem cells.

Olanzapine, an Atypical Antipsychotic, Inhibits Survivin Expression and Sensitizes Cancer Cells to Chemotherapeutic Agents

Olanzapine, an atypical antipsychotic, is now increasingly used as an off-label indication for the management of cancer patients with chemotherapy-induced nausea and vomiting (CINV). However, how olanzapine affects cancer cells per se remains poorly understood. The effects of olanzapine treatment and survivin knockdown, alone or in combination with chemotherapeutic agents, on survivin expression and cell viability were investigated in human cancer cell lines. Olanzapine reduced survivin expression in lung and pancreatic cancer stem cell (CSC) lines and sensitized them to chemotherapeutic agents such as 5-fluorouracil, gemcitabine, and cisplatin in a survivin expression-dependent manner. Olanzapine also reduced survivin expression and chemosensitized serum-cultured, non-CSC ovarian cancer cells that expressed survivin. Olanzapine may benefit cancer patients not only as an antiemetic for CINV, but also by enhancing the effects of chemotherapeutic agents through down-regulation of survivin, which has been implicated in multidrug chemoresistance.

Panaxynol, a natural Hsp90 inhibitor, effectively targets both lung cancer stem and non-stem cells

Cancer stem-like cells (CSCs) contribute to tumor recurrence and chemoresistance. Hence, strategies targeting CSCs are crucial for effective anticancer therapies. Here, we demonstrate the capacities of the non-saponin fraction of Panax ginseng and its active principle panaxynol to inhibit Hsp90 function and viability of both non-CSC and CSC populations of NSCLC in vitro and in vivo. Panaxynol inhibited the sphere forming ability of NSCLC CSCs at nanomolar concentrations, and micromolar concentrations of panaxynol suppressed the viability of NSCLC cells (non-CSCs) and their sublines carrying acquired chemoresistance with minimal effect on normal cells derived from various organs. Orally administered panaxynol significantly reduced lung tumorigenesis in KrasG12D/+ transgenic mice and mice carrying NSCLC xenografts without detectable toxicity. Mechanistically, panaxynol disrupted Hsp90 function by binding to the N-terminal and C-terminal ATP-binding pockets of Hsp90 without increasing Hsp70 expression. These data suggest the potential of panaxynol as a natural Hsp90 inhibitor targeting both the N-terminal and C-terminal of Hsp90 with limited toxicities.

RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer.

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer.

The mechanism of Sonic Hedgehog (Shh) pathway activation in non-small cell lung cancer (NSCLC) is poorly described. Using an antibody against the Shh C-terminal domain, we found a small population of Shh-positive (Shh+) cells in NSCLC cells. The objective of this study was to characterize these Shh+ cells. Shh+ and Shh- cells were sorted by using Fluorescence Activated Cell Sorting (FACS) on 12 commercial NSCLC cell lines. Functional analyses on sorted cells were performed with gene expression assays (qRT-PCR and microarray) and cells were treated with cytotoxic chemotherapy and a targeted inhibitor of Shh signaling (GDC0449). We used in vivo models of nude mice inoculated with Shh+ and Shh- sorted cells and drug-treated cells. Finally, we confirmed our results in fresh human NSCLC samples (n=48) paired with normal lung tissue. We found that Shh+ cells produced an uncleaved, full-length Shh protein detected on the membranes of these cells. Shh+ cells exerted a paracrine effect on Shh- cells, inducing their proliferation and migration. Shh+ cells were chemo-resistant and showed features of cancer stem cells (CSCs) in vitro and in vivo. Pharmacological inhibition of the Shh pathway suppressed their CSC features. A high percentage of Shh+ cells was associated with poor prognosis in early-stage NSCLC patients. In conclusion, we describe for the first time the presence of an abnormal membrane-bound full-length Shh protein in human cancer cells that allows the identification of CSCs in vitro and in vivo.

FGF1 and IGF1-conditioned 3D culture system promoted the amplification and cancer stemness of lung cancer cells

Lung cancer stem cells (LCSCs) are considered as the cellular origins of metastasis and relapse of lung cancer. However, routine two-dimensional culture system (2D-culture) hardly mimics the growth and functions of LCSCs in vivo and therefore significantly decreases the stemness activity of LCSCs. In this study, we constructed a special BME-based three-dimensional culture system (3D-culture) to amplify LCSCs in human lung adenocarcinoma cell line A549 cells and found 3D-culture promoted the enrichment and amplification of LCSCs in A549 cells displaying higher proliferation potential and invasion activity, but lower apoptosis. The expression and secretion levels of FGF1 and IGF1 were dramatically elevated in 3D-culture compared to 2D-culture. After growing in FGF1 and IGF1-conditioned 3D-culture, the proportion of LCSCs with specific stemness phenotypes in A549 cells significantly increased compared to that in conventional 3D suspension culture system. Further results indicated that FGF1 and IGF1 promoted the amplification and cancer stemness of LCSCs dependent on MAPK signaling pathway. Our data firstly established a growth factors-conditioned 3D-culture for LCSCs and demonstrated the effects of FGF1 and IGF1 in promoting the enrichment and amplification of LCSCs which might provide a feasible cell model in vitro for both mechanism study and translational research on lung cancer.