Expression Levels Of Stemness Markers Research Articles

TRIM28 suppresses cancer stem-like characteristics in gastric cancer cells through Wnt/β-catenin signaling pathways.

The influences of TRIM28 on the gastric tumorigenesis together with potential molecular mechanisms remain to be studied. We aimed at exploring the important effects of TRIM28 on gastric cancer (GC) and uncovering underling molecular mechanisms. Through immunohistochemistry analysis of 20 pairs of GC and the peritumoral tissues, the expression level of TRIM28 was determined. A variety of assays were applied to explore the important roles of TRIM28 in GC. Western blotting and qRT-PCR analyses were used to analyze the association between TRIM28 and the Wnt/β-catenin signaling pathway. TRIM28 was highly expressed in GC tissues than peritumoral tissues. And high expression level of TRIM28 in GC was associated with good prognostic effects. In vitro functional assays suggested TRIM28 knockdown enhanced the proliferation and clone formation of GC cell. Moreover, TRIM28 knockdown enhanced the expression level of stemness markers, strengthened sphere-forming and drug-resistance properties of GC cells, suggesting important effect on GC cell stemness. Besides, our analysis showed that the Wnt/β-catenin signaling was involved in the effect of TRIM28 on GC cell stemness property, and blocking Wnt/β-catenin signaling pathway obviously rescued the promotion influence of TRIM28 knockdown. Overall, TRIM28 has an important influence on regulating the stem-like property of GC cell via Wnt/β-catenin signaling, suggesting TRIM28 a promising drug target and a potential predictor of prognosis.

Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant hepatocellular carcinoma cells.

As a multiple tyrosine kinase inhibitor, sorafenib is widely used to treat hepatocellular carcinoma (HCC), but patients frequently face resistance problems. Because the mechanism controlling sorafenib-resistance is not well understood, this study focused on the connection between tumor characteristics and the Nrf2 signaling pathway in a sorafenib-resistant HCC cell line. A sorafenib-resistant HCC cell line (Huh7) was developed by increasing the dose of sorafenib in the culture medium until the target concentration was reached. Cell morphology, migration/invasion rates, and expression of stemness-related and ATP-binding cassette (ABC) transporter genes were compared between sorafenib-resistant Huh7 cells and parental Huh7 cells. Next, a small interfering RNA was used to knock down Nrf2 expression in sorafenib-resistant Huh7 cells, after which cell viability, stemness, migration, and ABC transporter gene expression were examined again. Proliferation, migration, and invasion rates of sorafenib-resistant Huh7 cells were significantly increased relative to the parental cells with or without sorafenib added to the medium. The expression levels of stemness markers and ABC transporter genes were up-regulated in sorafenib-resistant cells. After Nrf2 was knocked down in sorafenib-resistant cells, cell migration and invasion rates were reduced, and expression levels of stemness markers and ABC transporter genes were reduced. Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant HCC cells.

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations.

Human amniotic fluid stem cells (hAFSCs) possess some characteristics with mesenchymal stem cells (MSCs) and embryonic stem cells and have a broader differentiation potential compared to MSCs derived from other sources. Although hAFSCs are widely researched, their analysis mainly involves stem cells (SCs) obtained from normal, fetus-unaffected gestations. However, in clinical settings, knowledge about hAFSCs from normal gestations could be poorly translational, as hAFSCs from healthy and fetus-diseased gestations may differ in their differentiation and metabolic potential. Therefore, a more thorough investigation of hAFSCs derived from pathological gestations would provide researchers with the knowledge about the general characteristics of these cells that could be valuable for further scientific investigations and possible future clinical applicability. The goal of this study was to look into the neurogenic and metabolic potential of hAFSCs derived from diseased fetuses, when gestations were concomitant with polyhydramnios and compare them to hAFSCs derived from normal fetuses. Results demonstrated that these cells are similar in gene expression levels of stemness markers (SOX2, NANOG, LIN28A, etc.). However, they differ in expression of CD13, CD73, CD90, and CD105, as flow cytometry analysis revealed higher expression in hAFSCs from unaffected gestations. Furthermore, hAFSCs from “Normal” and “Pathology” groups were different in oxidative phosphorylation rate, as well as level of ATP and reactive oxygen species production. Although the secretion of neurotrophic factors BDNF and VEGF was of comparable degree, as evaluated with enzyme-linked immunosorbent assay (ELISA) test, hAFSCs from normal gestations were found to be more prone to neurogenic differentiation, compared to hAFSCs from polyhydramnios. Furthermore, hAFSCs from polyhydramnios were distinguished by higher secretion of pro-inflammatory cytokine TNFα, which was significantly downregulated in differentiated cells. Overall, these observations show that hAFSCs from pathological gestations with polyhydramnios differ in metabolic and inflammatory status and also possess lower neurogenic potential compared to hAFSCs from normal gestations. Therefore, further in vitro and in vivo studies are necessary to dissect the potential of hAFSCs from polyhydramnios in stem cell-based therapies. Future studies should also search for strategies that could improve the characteristics of hAFSCs derived from diseased fetuses in order for those cells to be successfully applied for regenerative medicine purposes.

Hyaluronic acid engrafted metformin loaded graphene oxide nanoparticle as CD44 targeted anti-cancer therapy for triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is the most aggressive form of breast cancer with limited treatment modalities. It is associated with high propensity of cancer recurrence. MethodsUV Spectroscopy, FTIR, DLS, Zeta potential, TEM and SEM were employed to characterize nanoparticles. MTT assay, Wound healing assay, SEM, Immunocytochemistry analysis, Western blot, RT-PCR, mammosphere formation assay were employed to study apoptosis, cell migration and stemness. Tumor regression was studied in chick embryo xenograft and BALB/c mice model. ResultsHylaluronic acid engrafted metformin loaded graphene oxide (HA-GO-Met) nanoparticles exhibited an anti-cancer efficacy at much lower dosage as compared to metformin alone. HA-GO-Met nanoparticles induced apoptosis and inhibited cell migration of TNBC cells by targeting miR-10b/PTEN axis via NFkB-p65. Upregulation of PTEN affected pAKT(473) expression that induced apoptosis. Cell migration was inhibited by reduction of pFAK/integrinβ1 expressions. Treatment inhibited epithelial mesenchymal transition (EMT) and reduced stemness as evident from the increase in E-cadherin expression, inhibition of mammosphere formation and low expression levels of stemness markers including nanog, oct4 and sox2 as compared to control. Moreover, tumor regression was studied in chick embryo xenograft and BALB/c mice model. HA-GO-Met nanoparticle treatment reduced tumor load and nullified toxicity in peripheral organs imparted by tumor. ConclusionsHA-GO-Met nanoparticles exhibited an enormous anti-cancer efficacy in TNBC in vitro and in vivo. General significanceHA-GO-Met nanoparticles induced apoptosis and attenuated cell migration in TNBC. It nullified overall toxicity imparted by tumor load. It inhibited EMT and reduced stemness and thereby addressed the issue of cancer recurrence.

Escherichia coli foster bladder cancer cell line progression via epithelial mesenchymal transition, stemness and metabolic reprogramming

Bacteria is recognized as opportunistic tumor inhabitant, giving rise to an environmental stress that may alter tumor microenvironment, which directs cancer behavior. In vitro infection of the T24 cell line with E. coli was performed to study the bacterial impact on bladder cancer cells. EMT markers were assessed using immunohistochemistry, western blot and RT-PCR. Stemness characteristics were monitored using RT-PCR. Furthermore, the metabolic reprograming was investigated by detection of ROS and metabolic markers. A significant (p ≤ 0.001) upregulation of vimentin as well as downregulation of CK19 transcription and protein levels was reported. A significant increase (p ≤ 0.001) in the expression level of stemness markers (CD44, NANOG, SOX2 and OCT4) was reported. ROS level was elevated, that led to a significant increase (p ≤ 0.001) in UCP2. This enhanced a significant increase (p ≤ 0.001) in PDK1 to significantly downregulate PDH (p ≤ 0.001) in order to block oxidative phosphorylation in favor of glycolysis. This resulted in a significant decrease (p ≤ 0.001) of AMPK, and a significant elevation (p ≤ 0.001) of MCT1 to export the produced lactate to extracellular matrix. Thus, bacteria may induce alteration to the heterogonous tumor cell population through EMT, CSCs and metabolic reprogramming, which may improve cancer cell ability to migrate and self-renew.

Anti-Breast Cancer Activities of Ketoprofen-RGD Conjugate by Targeting Breast Cancer Stem-Like Cells and Parental Cells.

Cancer Stem Cells (CSCs) play an important role in various stages of cancer development, advancement, and therapy resistance. Ketoprofen-RGD has been revealed to act as an anti-cancer agent against some tumors. We aimed to explore the effects of a novel Ketoprofen-RGD compound on the suppression of Breast Cancer Stem-like Cells (BCSCs) and their parental cells. Mammospheres were developed from MCF-7 cells and assessed by CSC surface markers through flowcytometry. The anti-proliferative and pro-apoptotic activities of Ketoprofen-RGD were measured by MTS assay and flowcytometry. The expression levels of stemness markers and JAK2/STAT proteins were measured by quantitative Real Time-PCR (qRT-PCR) and western blotting, respectively. Intracellular Reactive Oxygen Species (ROS) was measured using a cell permeable, oxidant-sensitive fluorescence probe (carboxy-H2DCFDA). Ketoprofen-RGD significantly reduced the mammosphere formation rate and the expression of three out of six stemness markers and remarkably decreased viability and induced apoptosis of spheroidal and parental cells compared to controls. Further experiments using CD95L, as a death ligand, and ZB4 antibody, as an extrinsic apoptotic pathway blocker, showed that Ketoprofen-RGD induced intrinsic pathway, suggesting a mechanism by which Ketoprofen-RGD triggers apoptosis. ROS production was also another way to induce apoptosis. Results of western blot analysis also revealed a marked diminish in the phosphorylation of JAK2 and STAT proteins. Our study, for the first time, elucidated an anti-BCSC activity for Ketoprofen-RGD via declining stemness markers, inducing toxicity, and apoptosis in these cells and parental cells. These findings may suggest this compound as a promising anti-breast cancer.

Abstract LB-020: The replication factor MCM10 maintains breast cancer stem-like cells that constitutively experience c-Myc-driven replication stress

Abstract Cancer stem-like cells (CSCs) are thought to be responsible for recurrence of tumors and poor prognosis due to their higher tumor-initiating capacity and resistance against chemotherapeutic agents, although the underlying mechanisms remain unclear. Here, we uncover a critical role of the replication factor mini-chromosome maintenance protein 10 (MCM10) in enabling breast cancer-derived CSCs to deal with constitutively accumulating DNA replication stress. Transcriptomic analysis of patient-derived breast cancer cells revealed that c-Myc downstream target genes and replication stress response genes were highly upregulated in CSC-enriched spheroid cells. Upregulated c-Myc enhanced the transcriptional activity especially at nucleoli, leading to DNA replication stress caused by collision of transcription and replication machineries. MCM10, one of the most upregulated factors in CSCs, showed important roles for enabling cells to survive under such condition through activating dormant origins. MCM10 depletion decreased tumorigenesis, tumor sphere-forming capacity, expression levels of stemness markers, and tumor-initiating capacity, suggesting that elevated MCM10 levels are vital for CSC survival and proliferation. On the other hand, overexpression of MCM10 helped cells to proliferate in medium containing hydroxyurea (HU), a chemotherapeutic agent that induces replication stress. We therefore conclude MCM10 plays critical roles for cancer cell proliferation under DNA replication stress, which is more pronounced in CSCs with upregulated c-Myc activity and in cells treated with chemotherapeutic agents, and propose to target MCM10 for therapy of both CSCs and other cancer cells. Citation Format: Noriko Gotoh. The replication factor MCM10 maintains breast cancer stem-like cells that constitutively experience c-Myc-driven replication stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-020.

Sevoflurane modulates the cancer stem cell-like properties and mitochondrial membrane potential of glioma via Ca2+-dependent CaMKII/JNK cascade

AimsGliomas are responsible for the majority of deaths from primary brain tumours. Sevoflurane showed inhibition effects on the tumor progression in vitro. However, whether sevoflurane could affect the stemness of glioma stem cells (GSCs) and the potential molecular mechanism have not been well elucidated. Main methodsEffects of sevoflurane on cell viability, proliferation and invasion ability of glioma cells as well as tumor growth in vivo were assessed. Sphere formation assay was performed to evaluate the effect of sevoflurane on the stemness of GSCs. Effects of sevoflurane on mitochondrial function was evaluated by intracellular/mitochondrial reactive oxygen species (ROS) level and mitochondrial membrane potential. Expression levels of proliferation-related proteins, stemness markers and proteins in CaMKII/JNK cascade were measured by Western blot. Key findingsSevoflurane inhibited the viability, proliferation and invasion ability of glioma cells (U87MG and U373MG). Western blot showed that sevoflurane decreased the expression levels of proliferation and invasion-related proteins. Sphere formation ability of GSCs, expression levels of stemness markers and mitochondrial function were significantly suppressed by sevoflurane. Moreover, sevoflurane treatment significantly increased the Ca2+ concentration and stimulated phosphorylation of CaMKII, JNK and IRS1. Ca2+ chelator BAPTA-AM combined with sevoflurane synergistically inhibited colony forming ability and the expression levels of proliferation-related proteins and stemness markers. In addition, the in vivo study further confirmed that sevoflurane inhibited tumor growth via Ca2+-dependent CaMKII/JNK cascade. SignificanceThe present study demonstrated that sevoflurane inhibited glioma tumorigenesis and modulated the cancer stem cell-like properties and mitochondrial membrane potential via activation of Ca2+-dependent CaMKII/JNK cascade.

Anti-Breast Cancer Activities of 8-Hydroxydaidzein by Targeting Breast Cancer Stem-Like Cells.

Cancer stem cells (CSCs) play an important role in various stages of cancer development and therapy refractoriness. 8-Hydroxydaidzein (8-OHD) has revealed anti-cancer activity in different tumors. Accordingly, we aimed to assess the effects of 8-OHD on the suppression of breast cancer stem-like cells (BCSCs). The anti-proliferative and pro-apoptotic properties of 8-OHD were examined by MTS assay and flowcytometry. The expression levels of stemness markers and JAK2/STAT proteins were measured by quantitative real time-PCR (qRT-PCR) and western blotting, respectively. 8-OHD significantly decreased three out of six stemness markers and remarkably reduced viability and induced apoptosis of spheroidal and parental cells compared to controls. Further experiments using CD95L, as a death ligand, and ZB4 antibody, as an extrinsic apoptotic pathway blocker, showed that 8-OHD induced apoptosis through the intrinsic pathway, proposing a mechanism by which 8-OHD triggers apoptosis. Results of western blot analysis also revealed a marked decline in the phosphorylation of JAK2 and STAT proteins. Our study, for the first time, elucidated an anti-BCSC activity for 8-OHD via decreasing stemness markers, inducing toxicity and stimulating apoptosis in these cells and parental cells. Our results also suggested a novel mechanism by which 8-OHD induces apoptosis in BCSCs.

Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration.

IntroductionHuman periodontal ligament mesenchymal stem cells (hPDLMSCs) have been known that they play important roles in homeostasis and regeneration of periodontal tissues. Additionally, spheroids are superior to monolayer-cultured cells. We investigated the characteristics and potential of periodontal tissue regeneration in co-cultured spheroids of hPDLMSCs and human umbilical vein endothelial cells (HUVECs) in vitro and in vivo.MethodsCo-cultured spheroids were prepared with cell ratios of hPDLMSCs: HUVECs = 1:1, 1:2, and 2:1, using microwell chips. Real-time polymerase chain reaction (PCR) analysis, Enzyme-Linked Immuno Sorbent Assay (ELISA), and nodule formation assay were performed to examine the properties of co-cultured spheroids. Periodontal tissue defects were prepared in the maxillary first molars of rats and subjected to transplantation assay.ResultsThe expression levels of stemness markers, vascular endothelial growth factor (VEGF), osteogenesis-related genes were up-regulated in co-cultured spheroids, compared with monolayer and spheroid-cultured hPDLMSCs. The nodule formation was also increased in co-cultured spheroids, compared with monolayer and spheroid cultures of hPDLMSCs. Treatment with co-cultured spheroids enhanced new cementum formation after 4 or 8 weeks of transplantation, although there was no significant difference in the new bone formation between co-cultured spheroids and hPDLMSC spheroids.ConclusionsWe found that co-cultured spheroids enhance the periodontal tissue regeneration. Co-cultured spheroids of hPDLMSCs and HUVECs may be a useful therapy that can induce periodontal tissue regeneration.

Pterostilbene Suppresses both Cancer Cells and Cancer Stem-Like Cells in Cervical Cancer with Superior Bioavailability to Resveratrol.

Increasing studies have reported that cancer stem cells (CSCs) play critical roles in therapeutic resistance, recurrence, and metastasis of tumors, including cervical cancer. Pterostilbene, a dimethylated derivative of resveratrol, is a plant polyphenol compound with potential chemopreventive activity. However, the therapeutic effect of pterostilbene against cervical CSCs remains unclear. In this study, we compared the anticancer effects of resveratrol and pterostilbene using both HeLa cervical cancer adherent and stem-like cells. Pterostilbene more effectively inhibited the growth and clonogenic survival, as well as metastatic ability of HeLa adherent cells than those of resveratrol. Moreover, the superior inhibitory effects of pterostilbene compared to resveratrol were associated with the enhanced activation of multiple mechanisms, including cell cycle arrest at S and G2/M phases, induction of ROS-mediated caspase-dependent apoptosis, and inhibition of matrix metalloproteinase (MMP)-2/-9 expression. Notably, pterostilbene exhibited a greater inhibitory effect on the tumorsphere-forming and migration abilities of HeLa cancer stem-like cells compared to resveratrol. This greater effect was achieved through more potent inhibition of the expression levels of stemness markers, such as CD133, Oct4, Sox2, and Nanog, as well as signal transducer and activator of transcription 3 signaling. These results suggest that pterostilbene might be a potential anticancer agent targeting both cancer cells and cancer stem-like cells of cervical cancer via the superior bioavailability to resveratrol.

Characterization of the metastatic potential of the floating cell component of MIA PaCa-2, a human pancreatic cancer cell line

In pancreatic cancer, morphologically and functionally heterogeneous cancer cells reside within the same patient. The heterogeneity is believed to promote metastasis and resistance to chemoradiotherapy. MIA PaCa-2, an established human pancreatic ductal adenocarcinoma (PDAC) cell line, contains round and spindle-shaped adherent cells, as well as, round floating cells. In this study, we aimed to assess if the floating cells might have greater metastatic potential and/or be more resistant to drug-induced apoptosis compared to adherent cells. Time-lapse analysis revealed that the two types of adherent cells transformed bilaterally, and some of the adherent, round cells converted to floating cells. Flow cytometry and electron microscopy showed that approximately 90% of the floating cells were viable. qRT-PCR analysis revealed that floating cells expressed lower levels of integrins and ATP-binding cassette (ABC) transporters than adherent cells. In contrast, except for vimentin, floating cells expressed more epithelial to mesenchymal transition markers than adherent cells. Floating cells included a larger population of G2/M-phase cells, and migration assays revealed a decreased migration ability by floating cells relative to adherent cells. A cell aggregation assay showed that the aggregative properties of the floating cells were lower than those of the adherent cells. In 3D culture, spheres derived from floating cells were more sensitive to anti-cancer drugs, including gemcitabine, 5-FU, and abraxane, than those derived from adherent cells. Expression levels of stemness markers in the spheres derived from floating cells were lower than those derived from adherent cells. Morphological characterization of human PDAC cell lines may help to clarify the series of alterations cancer cells undergo during the metastatic process and may contribute to the development of new PDAC diagnostics and more patient-specific treatments for those with PDAC.

EBV-miR-BART7-3p Imposes Stemness in Nasopharyngeal Carcinoma Cells by Suppressing SMAD7.

Cancer stem-like cells, possessing “stemness” properties, play crucial roles in progression, metastasis, and drug resistance in various cancers. Viral microRNAs (such as EBV-miR-BART7-3p), as exogenous regulators, have been discovered to regulate malignant progression of nasopharyngeal carcinoma (NPC), suggesting a possible role of viral microRNAs in imposing stemness. In this study, we found that EBV-miR-BART7-3p induce stemness of NPC cells. We firstly reported that EBV-miR-BART7-3p increased the percentage of side population cells, the development of tumor spheres, and the expression level of stemness markers in vitro. This viral microRNA also enhanced stem-like or cancer-initiating properties of NPC cells in vivo. Besides, we identified SMAD7 as a novel target gene of EBV-miR-BART7-3p in addition to PTEN gene we previously reported; this viral microRNA suppressed SMAD7, led to activation of TGF-β signaling, and eventually enhanced the stemness of NPC cells. Silencing of SMAD7 resembled the effects generated by EBV-miR-BART7-3p in NPC cells. After reconstitution of SMAD7, EBV-miR-BART7-3p-expressing cells underwent a phenotypic reversion. EBV-positive NPC cells were used to enable experimental validation. Finally, we further discovered that EBV-miR-BART7-3p increased chemo-resistance of NPC in vitro and in vivo, supporting that EBV-miR-BART7-3 resulted in increased stemness of NPC cells and lead to drug resistance and cancer recurrence. Overall, this study uncovered a novel mechanism underlying viral microRNA-associated stemness of NPC cells. This viral microRNA and its associated cellular genes may be potential therapeutic targets for restraining chemo-resistance and recurrence of NPC.

Matrix stiffness mediates stemness characteristics via activating the Yes-associated protein in colorectal cancer cells.

Matrix stiffness is an essential physical microenvironment in solid cancer. However, its influence on cancer stemness still remains elusive. Colorectal cancer (CRC) cell line HCT-116 was cultured in the matrix with various stiffness. The siYAP was applied to detect the changes of stemness markers. The cancer stemness markers, Yes-associated protein (YAP), Lamin A/C and downstream protein molecules, and their activation were measured after the treatment with anti-β1-integrin and FAK inhibitors. In CRC tissue samples, collagen deposition and the expression of α-SMA and CD133 were detected. The study found that the expression level of stemness markers and Lamin A/C increased as the matrix stiffness raised and was regulated by YAP activation in CRC stem cells. Inhibition of β1-integrin and FAK activation in a high stiffness cell culture medium significantly decreased the activation of YAP, PI3K, and AKT. Collagen was highly deposited in the CRC invasive tumor front (ITF), and the expression of CD133 was higher in ITF compared with normal tissue and the tumor cells. Moreover, the expression level of α-SMA was positively correlated with CD133 expression level. Together, our results suggest that activation of YAP in CRC plays an important role in the promotion of cancer stem cell properties by extracellular matrix stiffness in CRC.

Stemness and anti-cancer drug resistance in ATP-binding cassette subfamily G member 2 highly expressed pancreatic cancer is induced in 3D culture conditions.

The expression of ATP‐binding cassette subfamily G member 2 (ABCG2) is related to tumorigenic cancer stem cells (CSC) in several cancers. However, the effects of ABCG2 on CSC‐related malignant characteristics in pancreatic ductal adenocarcinoma (PDAC) are not well elucidated. In this study, we compared the characteristics of low (ABCG2−) and high (ABCG2+)‐ABCG2‐expressing PDAC cells after cell sorting. In adherent culture condition, human PDAC cells, PANC‐1, contained approximately 10% ABCG2+ cell populations, and ABCG2+ cells displayed more and longer microvilli compared with ABCG2− cells. Unexpectedly, ABCG2+ cells did not show significant drug resistance against fluorouracil, gemcitabine and vincristine, and ABCG2− cells exhibited higher sphere formation ability and stemness marker expression than those of ABCG2+ cells. Cell growth and motility was greater in ABCG2− cells compared with ABCG2+ cells. In contrast, epithelial‐mesenchymal transition ability between ABCG2− and ABCG2+ cells was comparable. In 3D culture conditions, spheres derived from ABCG2− cells generated a large number of ABCG2+ cells, and the expression levels of stemness markers in these spheres were higher than spheres from ABCG2+ cells. Furthermore, spheres containing large populations of ABCG2+ cells exhibited high resistance against anti‐cancer drugs presumably depending on ABCG2. ABCG2+ cells in PDAC in adherent culture are not correlated with stemness and malignant behaviors, but ABCG2+ cells derived from ABCG2− cells after sphere formation have stemness characteristics and anti‐cancer drug resistance. These findings suggest that ABCG2− cells generate ABCG2+ cells and the malignant potential of ABCG2+ cells in PDAC varies depending on their environments.

The Endogenous GRP78 Interactome in Human Head and Neck Cancers: A Deterministic Role of Cell Surface GRP78 in Cancer Stemness

Cell surface glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, was suggested to be a cancer stem cell marker, but the influence of this molecule on cancer stemness is poorly characterized. In this study, we developed a mass spectrometry platform to detect the endogenous interactome of GRP78 and investigated its role in cancer stemness. The interactome results showed that cell surface GRP78 associates with multiple molecules. The influence of cell population heterogeneity of head and neck cancer cell lines (OECM1, FaDu, and BM2) according to the cell surface expression levels of GRP78 and the GRP78 interactome protein, Progranulin, was investigated. The four sorted cell groups exhibited distinct cell cycle distributions, asymmetric/symmetric cell divisions, and different relative expression levels of stemness markers. Our results demonstrate that cell surface GRP78 promotes cancer stemness, whereas drives cells toward a non-stemlike phenotype when it chaperones Progranulin. We conclude that cell surface GRP78 is a chaperone exerting a deterministic influence on cancer stemness.

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.

Reversion of malignant phenotypes of human glioblastoma cells by β-elemene through β-catenin-mediated regulation of stemness-, differentiation- and epithelial-to-mesenchymal transition-related molecules.

BackgroundGlioblastoma is the most common and lethal type of primary brain tumor. β-Elemene, a natural plant drug extracted from Curcuma wenyujin, has shown strong anti-tumor effects in various tumors with low toxicity. However, the effects of β-elemene on malignant phenotypes of human glioblastoma cells remain to be elucidated. Here we evaluated the effects of β-elemene on cell proliferation, survival, stemness, differentiation and the epithelial-to-mesenchymal transition (EMT) in vitro and in vivo, and investigated the mechanisms underlying these effects.MethodsHuman primary and U87 glioblastoma cells were treated with β-elemene, cell viability was measured using a cell counting kit-8 assay, and treated cells were evaluated by flow cytometry. Western blot analysis was carried out to determine the expression levels of stemness markers, differentiation-related molecules and EMT-related effectors. Transwell assays were performed to further determine EMT of glioblastoma cells. To evaluate the effect of β-elemene on glioblastoma in vivo, we subcutaneously injected glioblastoma cells into the flank of nude mice and then intraperitoneally injected NaCl or β-elemene. The tumor xenograft volumes were measured every 3 days and the expression of stemness-, differentiation- and EMT-related effectors was determined by Western blot assays in xenografts.Resultsβ-Elemene inhibited proliferation, promoted apoptosis, impaired invasiveness in glioblastoma cells and suppressed the growth of animal xenografts. The expression levels of the stemness markers CD133 and ATP-binding cassette subfamily G member 2 as well as the mesenchymal markers N-cadherin and β-catenin were significantly downregulated, whereas the expression levels of the differentiation-related effectors glial fibrillary acidic protein, Notch1, and sonic hedgehog as well as the epithelial marker E-cadherin were upregulated by β-elemene in vitro and in vivo. Interestingly, the expression of vimentin was increased by β-elemene in vitro; this result was opposite that for the in vivo procedure. Inhibiting β-catenin enhanced the anti-proliferative, EMT-inhibitory and specific marker expression-regulatory effects of β-elemene.Conclusionsβ-Elemene reversed malignant phenotypes of human glioblastoma cells through β-catenin-involved regulation of stemness-, differentiation- and EMT-related molecules. β-Elemene represents a potentially valuable agent for glioblastoma therapy.

In vitro evaluation of different methods of handling human liposuction aspirate and their effect on adipocytes and adipose derived stem cells.

Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue-derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10-20-30 min) with the centrifugation technique at different speeds (90-400-1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, "middle layer", and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45-) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available.

Phenotypic and functional characterization of long-term cryopreserved human adipose-derived stem cells.

Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.