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The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities., The authors barcoded glioma-initiating cells (GIC) using combinations of virally encoded fluorophores. GIC show a strong tendency to form clonal populations over as few as two passages. Combined with stem cell marker phenotyping and computational analysis, they could trace the fate of such populations. This model presents an approach for rapid assessment of newly established GIC to assess tumour-specific vulnerabilities.

Glioblastoma (GBM) is a heterogeneous tumor of the brain with a poor prognosis due to recurrence and drug resistance following therapy. Genome-wide profiling has revealed the existence of distinct GBM molecular subtypes that respond differently to aggressive therapies. Despite this, molecular subtype does not predict recurrence or drug resistance and overall survival is similar across subtypes. One of the key features contributing to tumor recurrence and resistance to therapy is proposed to be an underlying subpopulation of resistant glioma stem cells (GSC). CD133 expression has been used as a marker of GSCs, however recent evidence suggests the relationship between CD133 expression, GSCs and molecular subtype is more complex than initially proposed. The expression of CD133, Olig2 and CD44 was investigated using patient derived glioma stem-like cells (PDGCs) in vitro and in vivo. Different PDGCs exhibited a characteristic equilibrium of distinct CD133+ and CD44+ subpopulations and the influence of environmental factors on the intra-tumor equilibrium of CD133+ and CD44+ cells in PDGCs was also investigated, with hypoxia inducing a CD44+ to CD133+ shift and chemo-radiotherapy inducing a CD133+ to CD44+ shift. These data suggest that surveillance and modulation of intra-tumor heterogeneity using molecular markers at initial surgery and surgery for recurrent GBM may be important for more effective management of GBM. ispartof: PLoS One vol:12 issue:2 pages:e0172791- ispartof: location:United States status: published

BACKGROUND/AIM Oral squamous cell carcinoma (OSCC) is a highly invasive malignancy with poor prognosis. Recent reports suggest that Sonic Hedgehog (SHH) plays a key role in tumor progression and worsens the response to therapy, possibly through an association with a cancer stem cell (CSC) phenotype. The objective of our study was to investigate the relationship between SHH expression and CSC markers in OSCC. MATERIALS AND METHODS A total of 67 OSCC specimens were immunostained for SHH and CSC markers using specific antibodies and expression was correlated with clinicopathological parameters. RESULTS SHH expression was significantly correlated with CD133 (p=0.026, r=0.272) and SRY-box transcription factor 2 (SOX2; p

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro. To determine whether Prom1-expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo. Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion. SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

Purpose The identification of prognostically and therapeutically relevant molecular markers is fundamental to the further development of personalised therapies in brain tumours. Current therapeutic options for the treatment of gliomas rely mainly on surgical resection and the inhibition of tumour cell proliferation by irradiation and chemotherapy. Glioma stem cells are a subpopulation of proliferating tumour cells that have self-renewal capacity and can give rise to heterogeneous cells that comprise the tumour and are thought to play a role in the resistance of gliomas to therapy. The aim of this study was to evaluate the expression of markers of glioma stem cells and differentiated glial cells in proliferating glioma cells in comparison to the overall expression of the respective markers in the tumour tissue. Methods Tissue microarrays were assembled from specimen of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, and anaplastic ependymoma. These were immunohistochemically double stained with antibodies against the proliferation-associated antigen Ki67 and marker proteins for glioma stem cells (CD133, Nestin, Musashi, CD15, CD44), and differentiated glioma cells (GFAP, MAP2c). Results The expression of both glial and glioma stem cell markers differs between proliferating and non-proliferating glioma cells. Furthermore, the proliferating cells in the different glial tumour entities show a different expression profile. Conclusion Further analysis of marker expression in proliferating glioma cells and correlation with clinical outcome and susceptibility to irradiation and chemotherapy might help establish new biomarkers and therapies for glioma.

Tumor heterogeneity is one of the ongoing huddles in the field of colon cancer therapy. It is evident that there are countless clones which exhibit different phenotypes and therefore, single cell analysis is inevitable. Cancer stem cells (CSCs) are rare cell population within tumor which is known to function in cancer metastasis and recurrence. Although there have been trials to prove intra-tumoral heterogeneity using single cell sequencing, that of CSCs has not been clearly elucidated. Here, we articulate the presence of heterogeneous subclones within CD133 positive cancer stem cells through single cell sequencing. As a proof of principle, we performed phenotype-based high-throughput laser isolation and single cell sequencing (PHLI-seq) of CD133 positive cells in a frozen tumor tissue obtained from a patient with colorectal cancer. The result proved that CD133 positive cells were shown to be heterogeneous both in copy number and mutational profiles. Single cancer stem cell specific mutations such as RNF144A, PAK2, PARP4, ADAM21, HYDIN, KRT38 and CELSR1 could be also detected in liver metastatic tumor of the same patient. Collectively, these data suggest that single cell analysis used to spot subclones with genetic variation within rare population, will lead to new strategies to tackle colon cancer metastasis.

Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis., Human ribonuclease 1 (hRNase 1) regulates innate immunity, hemostasis and RNA clearance. Here, the authors report an alternative function of hRNase 1 as a secretory ligand of Eph receptor EphA4 to enhance breast cancer stemness and promote breast tumour initiation.

Cancer stem cells (CSCs) represent a small subpopulation of cells found within tumors that exhibit properties of self-renewal, like normal stem cells. CSCs have been defined as a crucial factor involved in driving cancer relapse, chemoresistance and metastasis. Prominin-1 (CD133) is one of the most well-characterized markers of CSCs in various tumor types, including hepatocellular carcinoma (HCC). CD133+ cells have been demonstrated to be involved in metastasis, tumorigenesis, tumor recurrence, and resistance to treatment in HCC. CD133-related clinical prognosis prediction, and targeted therapy have highlighted the clinical significance of CD133 in HCC. However, there remains controversy over the role of CD133 in experimental and clinical research involving HCC. In this article, we summarize the fundamental cell biology of CD133 in HCC cells and discuss the important characteristics of CD133+ in HCC cells. Furthermore, the prognostic value of CD133, and therapeutic strategies for its targeting in HCC, is also reviewed.

Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH) signal regulates stem cells in normal prostate epithelium by affecting cell behavior, survival, proliferation, and maintenance. Aberrant SHH pathway activation leads to an unsuitable expansion of stem cell lineages in the prostate epithelium and the transformation of prostate CSCs (PCSCs). Zoledronic acid (ZOL), one of the third-generation bisphosphonates, effectively prevented bone metastasis and treated advanced prostate cancer despite androgen deprivation therapy. Despite strong evidence for the involvement of the SHH in human PCSCs survival and drug resistance, the roles of SHH in the PCSCs-related resistance to ZOL remain to be fully elucidated. The present study aimed to investigate the role of the SHH pathway in ZOL resistance of PCSCs in 2D and three 3D cell culture conditions. For this purpose, we isolated CD133(high)/ CD44(high) PCSCs using a flow cytometer. Following ZOL treatment, mRNA and protein expressions of the components of the SHH signaling pathway in PCSCs and non-CSCs were analyzed using qRT-PCR and Immunofluorescence staining, respectively. Our finding suggested that SHH signaling may be activated by different mechanisms that lead to avoidance of the inhibition effect of ZOL. Thereby, SHH pathways may be associated with the resistance to ZOL developed by prostate CSCs. Inhibition of CSCs-related SHH signaling along with ZOL treatment should be considered to achieve improvement in survival or delayed treatment failure and prevention of the CSCs-related drug resistance., Ege University Research Foundation [2016-TIP-079], This work was supported by Ege University Research Foundation (Project no: 2016-TIP-079).

High-dose acetaminophen (AAP) with delayed rescue using n-acetylcysteine (NAC), the FDA-approved antidote to AAP overdose, has demonstrated promising antitumor efficacy in early phase clinical trials. However, the mechanism of action (MOA) of AAP's anticancer effects remains elusive. Using clinically relevant AAP concentrations, we evaluated cancer stem cell (CSC) phenotype in vitro and in vivo in lung cancer and melanoma cells with diverse driver mutations. Associated mechanisms were also studied. Our results demonstrated that AAP inhibited 3D spheroid formation, self-renewal, and expression of CSC markers when human cancer cells were grown in serum-free CSC media. Similarly, anti-CSC activity was demonstrated in vivo in xenograft models - tumor formation following in vitro treatment and ex-vivo spheroid formation following in vivo treatment. Intriguingly, NAC, used to mitigate AAP's liver toxicity, did not rescue cells from AAP's anti-CSC effects, and AAP failed to reduce glutathione levels in tumor xenograft in contrast to mice liver tissue suggesting nonglutathione-related MOA. In fact, AAP mediates its anti-CSC effect via inhibition of STAT3. AAP directly binds to STAT3 with an affinity in the low micromolar range and a high degree of specificity for STAT3 relative to STAT1. These findings have high immediate translational significance concerning advancing AAP with NAC rescue to selectively rescue hepatotoxicity while inhibiting CSCs. The novel mechanism of selective STAT3 inhibition has implications for developing rational anticancer combinations and better patient selection (predictive biomarkers) for clinical studies and developing novel selective STAT3 inhibitors using AAP's molecular scaffold.

Glioblastoma (GBM) is the most common and deadliest tumor of the central nervous system. GBM has poor prognosis and glioma stem cells (GSCs) are implicated in tumor initiation and therapy resistance. Estrogen receptor β (ERβ) is expressed in GBM and exhibit tumor suppressive function. However, the role of ERβ in GSCs and the therapeutic potential of ERβ agonists on GSCs remain largely unknown. Here, we examined whether ERβ modulates GSCs stemness and tested the utility of two ERβ selective agonists (LY500307 and Liquiritigenin) to reduce the stemness of GSCs. The efficacy of ERβ agonists was examined on GSCs isolated from established and patient derived GBMs. Our results suggested that knockout of ERβ increased the proportion of CD133+ and SSEA+ positive GSCs and overexpression of ERβ reduced the proportion of GSCs in GBM cells. Overexpression of ERβ or treatment with ERβ agonists significantly inhibited the GSCs cell viability, neurosphere formation, self-renewal ability, induced the apoptosis and reduced expression of stemness markers in GSCs. RNA sequencing analysis revealed that ERβ agonist modulate pathways related to stemness, differentiation and apoptosis. Mechanistic studies showed that ERβ overexpression or agonist treatment reduced glutamate receptor signaling pathway and induced apoptotic pathways. In orthotopic models, ERβ overexpression or ERβ agonists treatment significantly reduced the GSCs mediated tumor growth and improved the mice overall survival. Immunohistochemical studies demonstrated that ERβ overexpression decreased SOX2 and GRM3 expression and increased expression of GFAP in tumors. These results suggest that ERβ activation could be a promising therapeutic strategy to eradicate GSCs. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Glioma stem cells (GSCs) are implicated in GBM development, progression and chemo/radiation resistance, and their elimination is essential for the development of effective therapies. This study shows that ERβ activation by agonists or upregulation of its expression, reduced viability, self-renewal and increased apoptosis in GSCs. Mechanistically, ERβ activation reduced glutamate receptor signaling in GSCs, reduced the tumor progression and improved the survival in orthotopic murine models. This study proposes that ERβ agonists may represent promising therapeutic agents for eliminating GSCs, and thereby improving the overall survival of GBM patients.

Background/aim CD44 and CD133 have been implicated as biomarkers of cancer cells and their expression could be analyzed to identify circulating tumor cells. Although CD44 and CD133 have been shown to be expressed in prostate cancer cells, a differential expression pattern has been reported depending on the tumor stage and cell line examined. We further investigated CD44 and CD133 expression in different prostate cancer cell lines to confirm whether their expression is distinguishable among patients with various tumor stages. Materials and methods CWR22Rv1, PC3, LNCaP, and DU145 cell lines were cultured and the cell morphology was observed for three days. The single expression of CD44 or CD133 and their combined expression were analyzed by flow cytometry. Results We report that the single expression of CD133 was less than 5% in all cell lines examined here. PC3 and DU145 cells displayed a high expression of CD44 (>93%), while the expression of CD44 was less than 4% in CWR22Rv1 and LNCaP cells. CWR22Rv1 was the only cell line that demonstrated a high co-expression of both CD44 and CD133. Conclusion Both single and combined expression of CD44 and CD133 should be considered when validating the detection of prostate cancer cells in circulating tumor cells.

Glioblastoma (GBM) is an aggressive malignant brain tumor; surgery, radiation, and temozolomide still remain the main treatments. There is evidence that E2F1 is overexpressed in various types of cancer, including GBM. E2F1 is a transcription factor that controls the cell cycle progression and regulates DNA damage responses and the proliferation of pluripotent and neural stem cells. To test the potentiality of E2F1 as molecular target for GBM treatment, we suppressed the E2F1 gene (siRNA) in the U87MG cell line, aiming to inhibit cellular proliferation and modulate the radioresistance of these cells. Following E2F1 suppression, associated or not with gamma-irradiation, several assays (cell proliferation, cell cycle analysis, neurosphere counting, and protein expression) were performed in U87MG cells grown as monolayer or neurospheres. We found that siE2F1-suppressed cells showed reduced cell proliferation and increased cell death (sub-G1 fraction) in monolayer cultures, and also a significant reduction in the number of neurospheres. In addition, in irradiated cells, E2F1 suppression caused similar effects, with reduction of the number of neurospheres and neurosphere cell numbers relative to controls; these results suggest that E2F1 plays a role in the maintenance of GBM stem cells, and our results obtained in neurospheres are relevant within the context of radiation resistance. Furthermore, E2F1 suppression inhibited or delayed GBM cell differentiation by maintaining a reasonable proportion of CD133+ cells when grown at differentiation condition. Therefore, E2F1 proved to be an interesting molecular target for therapeutic intervention in U87MG cells.

Hepatocellular carcinoma (HCC) is one of the leading malignant diseases worldwide, but therapeutic targets for HCC are lacking. Here, we characterized a significant upregulation of Small Nuclear Ribonucleoprotein Polypeptides B and B1 (SNRPB) in HCC via qRT-PCR, western blotting, tissue microarray and public database analyses. Increased SNRPB expression was positively associated with adjacent organ invasion, tumor size, serum AFP level and poor HCC patient survival. Next, we transfected SNRPB into HCC cells to construct SNRPB-overexpressing cell lines, and short hairpin RNA targeting SNRPB was used to silence SNRPB in HCC cells. Functional studies showed that SNRPB overexpression could promote HCC cell malignant proliferation and stemness maintenance. Inversely, SNRPB knockdown in HCC cells caused inverse effects. Importantly, analysis of alternative splicing by RNA sequencing revealed that SNRPB promoted the formation of AKT3-204 and LDHA-220 splice variants, which activated the Akt pathway and aerobic glycolysis in HCC cells. In conclusion, SNRPB could serve as a prognostic predictor for patients with HCC, and it promotes HCC progression by inducing metabolic reprogramming.

Adult progenitor cell populations typically exist in a quiescent state within a controlled niche environment. However, various stresses or forms of damage can disrupt this state, which often leads to dysfunction and aging. We built a glucocorticoid (GC)-induced liver damage model of mice, found that GC stress induced liver damage, leading to consequences for progenitor cells expansion. However, the mechanisms by which niche factors cause progenitor cells proliferation are largely unknown. We demonstrate that, within the liver progenitor cells niche, Galectin-3 (Gal-3) is responsible for driving a subset of progenitor cells to break quiescence. We show that GC stress causes aging of the niche, which induces the up-regulation of Gal-3. The increased Gal-3 population increasingly interacts with the progenitor cell marker CD133, which triggers focal adhesion kinase (FAK)/AMP-activated kinase (AMPK) signaling. This results in the loss of quiescence and leads to the eventual stemness exhaustion of progenitor cells. Conversely, blocking Gal-3 with the inhibitor TD139 prevents the loss of stemness and improves liver function. These experiments identify a stress-dependent change in progenitor cell niche that directly influence liver progenitor cell quiescence and function.

Thyroid stimulating hormone deficiency is the cornerstone of treatment for metastatic thyroid cancer. Due to the loss of follicular epithelial cells in thyroid cancer, the thyroid gland degenerates to 85% of its original size. When thyroid stimulating hormone is restored, follicular epithelial cells in thyroid cancer regenerate, which is postulated to be related to stem-like cells. By single cell RNA seq, we found a group of rare thyroid follicular epithelial cells in mouse metastatic thyroid cancer, which expressed stem-like genes (CD44V6+ and CD133+) and a large number of differentiated cells (CD44V6+ and CD24+). In mouse and in organoids, the two subsets contribute equally to metastatic thyroid cancer regeneration. The analysis of human metastatic thyroid cancer revealed that the differentiated thyroid follicular epithelial cell subpopulation was similar to that of the stem like epithelial cell subpopulation, and the regeneration potential was also enhanced after thyroid stimulating hormone ablation. Accordingly, we propose that the regeneration of metastatic thyroid cancer is driven by almost all persistent thyroid follicular epithelial cells, not only by few stem-like cells.

We enriched a specific subpopulation of liver cancer stem cells using multiple surface markers and then cultured the cells with modified chemically defined medium to maintain the cancer stem cell properties for 2 weeks in vitro. This study may provide an effective in vitro model for the study of the biological properties of liver cancer stem cells and their use in targeted drug screening., Liver cancer stem cells (L‐CSCs) are considered to be an important therapeutic target for hepatocellular carcinoma (HCC). This study provides a new in vitro long‐term culture model for a specific subpopulation of L‐CSCs enriched by cell surface markers. We combined CD13, CD133 and EpCAM to selectively enrich L‐CSCs, which we then cultured in modified chemically defined medium. The enriched L‐CSCs exhibited enhanced proliferation, self‐renewal and long‐term clonal maintenance ability as compared with non‐CSCs. Compared with wild‐type hepatocellular carcinoma, the expression of stemness surface markers, oncogenes, drug resistance and tumorigenicity in enriched L‐CSCs was significantly increased. In summary, the subpopulation of L‐CSCs still maintains cancer stem cell‐related phenotypes after 14 days of culture.

Melanoma stem cells (MSCs) are characterized by their unique cell surface proteins and aberrant signaling pathways. These stemness properties are either in a causal or consequential relationship to melanoma progression, treatment resistance and recurrence. The functional analysis of CD133+ and CD133- cells in vitro and in vivo revealed that melanoma progression and treatment resistance are the consequences of CD133 signal to PI3K pathway. CD133 signal to PI3K pathway drives two downstream pathways, the PI3K/Akt/MDM2 and the PI3K/Akt/MKP-1 pathways. Activation of PI3K/Akt/MDM2 pathway results in the destabilization of p53 protein, while the activation of PI3K/Akt/MKP-1 pathway results in the inhibition of mitogen-activated protein kinases (MAPKs) JNK and p38. Activation of both pathways leads to the inhibition of fotemustine-induced apoptosis. Thus, the disruption of CD133 signal to PI3K pathway is essential to overcome Melanoma resistance to fotemustine. The pre-clinical verification of in vitro data using xenograft mouse model of MSCs confirmed the clinical relevance of CD133 signal as a therapeutic target for melanoma treatment. In conclusion, our study provides an insight into the mechanisms regulating MSCs growth and chemo-resistance and suggested a clinically relevant approach for melanoma treatment.

The population of CD133 positive cancer cells has been reported to be responsible for drug resistance of hepatocellular carcinoma (HCC). However, the potential molecular mechanism by which CD133+ HCC cells develop drug resistance is still unclear. In this study, we found that CD133+ HepG2 and Huh7 cells were resistant to cisplatin treatment, compared to the CD133- HepG2 and Huh7 cells. However, treatment with osthole, a natural coumarin isolated from umbelliferae plant monomers, was found to resensitize CD133+ HepG2 and Huh7 cells to cisplatin treatment. In the mechanism research, we found that treatment with osthole increased the expression of PTEN. As a result, osthole inhibited the phosphorylation of AKT and Bad to decrease the amount of free Bcl-2 in CD133+ HepG2 and Huh7 cells. Finally, cisplatin-induced mitochondrial apoptosis was expanded. In conclusion, combination treatment with osthole can resensitize CD133+ HCC cells to cisplatin treatment via the PTEN/AKT pathway.

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.

Background Although thrombospondins 4 (THBS4) participates in controlling the biology of prostate cancer (PCa), the mechanism underlying this regulation remains unknown. Hence, this study aims to identify the regulatory effects of THBS4 on the PCa stem cell-like properties and the potential mechanism associated with the phosphatidylinositol 3'-kinase (PI3K)/protein kinase B (Akt) pathway. Methods PCa stem cells were sorted and identified using flow cytometry and THBS4 expression in the identified PCa stem cells was measured using Western blot assay. THBS4 was overexpressed or silenced in PCa stem cells, following which, self-renewal, proliferation, cell cycle distribution, and apoptosis of PCa stem cells were assessed as well as tumorigenicity in vivo was evaluated. PI3K/Akt pathway inhibitor was applied to identify its involvement in the regulatory roles of THBS4 in PCa stem cells. Results THBS4 was expressed at a higher level in PCa stem cells than in PCa cells. The overexpression of THBS4 promoted the self-renewal and proliferation, curbed the apoptosis of PCa stem cells, and enhanced the in vivo tumorigenicity, which was achieved by activating the PI3K/Akt pathway. On the contrary, short-hairpin RNA-mediated silencing of THBS4 exhibited suppressive effects on those cancer stem cell (CSC)-like properties and promotive effects on their apoptosis. Conclusion THBS4 silencing can impede the CSC-like properties in PCa via blockade of the PI3K/Akt pathway, which provides patients with PCa a new therapeutic target.

Background Adaptive drug resistance is an unfavourable prognostic factor in cancer therapy. Pemetrexed-resistant lung cancer cells possess high-metastatic ability via ERK–ZEB1 pathway-activated epithelial–mesenchymal transition. GMI is a fungal immunomodulatory protein that suppresses the survival of several cancer cells. Methods Cell viability was analysed by MTT, clonogenic, tumour spheroid, and cancer stem cell sphere assays. Western blot assay was performed to detect the protein expression. Chemical inhibitors and ATG5 shRNA were used to inhibit autophagy. Tumour growth was investigated using xenograft mouse model. Results GMI decreased the viability with short- and long-term effects and induced autophagy but not apoptosis in A549/A400 cells. GMI downregulated the expression levels of CD133, CD44, NANOG and OCT4. GMI induces the protein degradation of CD133 via autophagy. CD133 silencing decreased the survival and proliferation of A549/A400 cells. GMI suppressed the growth and CD133 expression of A549/A400 xenograft tumour. Conclusions This study is the first to reveal the novel function of GMI in eliciting cytotoxic effect and inhibiting CD133 expression in pemetrexed-resistant lung cancer cells via autophagy. Our finding provides evidence that CD133 is a potential target for cancer therapy.

Altres ajuts: Acknowledgements TAM, LG, NTC, I-JL, RT, JRH, and SR were funded by Medical Research Council (MRC, UK) Molecular Haematology Unit grant MC_UU_12009/6, MC_UU_00016/6, and MR/ M003221/1. AR was supported by a Bloodwise Clinician Scientist Fellowship (grants: 14041 and 17001), Wellcome Trust Clinical Research Career Development Fellowship (216632/Z/19/Z), Lady Tata Memorial International Fellowship, and EHA-ASH Translational Research Training in Hematology Fellowship. SOB was funded by the Department of Paediatrics and Alexander Thatte Fund, University of Oxford. DJHFK is funded by a CIHR Postdoctoral Fellowship. IR is supported by the NIHR Oxford BRC, by a Bloodwise Program Grant (13001) and by the MRC Molecular Haematology Unit (MC_UU_12009/14). PV via the Molecular Haematology Unit (MC_UU_12009) and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC) Programme. We would like to acknowledge the WIMM Flow Cytometry Facility which is supported by the MRC HIU, MRC MHU (MC_UU_12009), NIHR Oxford BRC, Kay Kendall Leukemia Fund (KKL1057), John Fell Fund (131/030 and 101/517), the EPA fund (CF182 and CF170), and by the WIMM Strategic Alliance awards G0902418 and MC_UU_12025. We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust Grant Reference 090532/Z/09/Z); the MRC WIMM Centre for Computational Biology (CCB), Radcliffe Department of Medicine, University of Oxford; and Jelena Telenius for the use of her pipelines. B-ALL work in PM's lab is financially supported by [...], the Fundación Uno entre Cienmil, the Fundación Leo Messi. PM also acknowledges structural support from the Obra Social La Caixa-Fundaciò Josep Carreras. The human fetal material was provided by the Joint MRC/ Wellcome Trust Grant 099175/Z/ 12/Z Human Developmental Biology Resource (http://hdbr.org). We gratefully acknowledge the kind generosity of patients, their parents, and staff at Great Ormond Street Hospital, London. MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.

Cluster of differentiation 133 (CD133) is a transmembrane glycoprotein that has been reported as a marker of cancer stem cells or cancer-initiating cells in various cancers. However, its contribution to tumorigenesis and differentiation remains to be elucidated. To determine the role of CD133 in colon cancer, we silenced CD133 in human colon cancer cells. Silencing of CD133 results in decreased cell proliferation, survival, migration, invasion, and glucose transport. These effects are mediated by downregulation of the human epidermal growth factor receptor 3 (HER3)/Akt/mTOR signaling pathway, culminating in reduced expression of the glucose transporter GLUT1. We also confirm that the cellular phenotypes of CD133-silenced cells are mediated by GLUT1 downregulation. We conclude that CD133 is a potential tumor initiator that positively regulates GLUT1 expression through modulation of HER3/Akt/mTOR signaling.

Digestive system cancers are the most frequent cancers worldwide and often associated with poor prognosis because of their invasive and metastatic characteristics. Recent studies have found that the plasticity of cancer cells can impart cancer stem-like properties via the epithelial-mesenchymal transition (EMT). Cancer stem-like properties such as tumor initiation are integral to the formation of metastasis, which is the main cause of poor prognosis. Numerous markers of cancer stem cells (CSCs) have been identified in many types of cancer. Therefore, CSCs, via their stem cell-like functions, may play an important role in prognosis after surgery. While several reports have described prognostic analysis using CSC markers, few reviews have summarized CSCs and their association with prognosis. Herein, we review the prognostic potential of eight CSC markers, CD133, CD44, CD90, ALDH1A1, EPCAM, SOX2, SOX9, and LGR5, in digestive cancers including those of the pancreas, colon, liver, gastric, and esophagus.

There is a diverse population of stem cells in human breast milk that can be employed for therapeutic purposes as a reservoir of cells. The current study mainly aimed to determine the nature markers expressing on stem cells. For this aim, the expression of embryonic stem cell markers, as well as the expression of endothelial, mesenchymal, neural, and hematopoietic markers were evaluated by the flow cytometry analysis in fresh colostrum, breast milk, and cultured colostrum samples. The results showed that the embryonic (OCT4, SOX2, HLA-DR), hematopoietic (CD33, CD45, CD117), neural (CD133, Nestin), and mesenchymal (CD44, SCA1) stem cell markers present in colostrum had higher expression in comparison with their counterpart markers in fresh breast milk. The expression markers of stem cells in colostrum following a 2-week culture period were significantly increased compared with their counterpart markers in colostrum before the culture process. In the culture of breastmilk, cells were not observed adherent cells and colonies. Our findings form flow cytometry and cell culture suggest that the lactation stage could be one of the factors influencing the stem cell population and, consequently, the cultivation of breastmilk cells. The present study indicates that colostrum is a tremendous source of stem cells that could be applied in cell-based research.

HuH-7 cells, derived from human hepatocarcinoma, are known to contain the CD133-positive cancer stem cell populations. HuH-7 cells showed higher ATP synthesis activity through the respiratory chain compared to another human hepatocarcinoma cell line HepG2 and showed an especially higher glycerol-3-phosphate (G3P)-driven ATP synthesis (G3P-ATPase) activity. We found that the CD133-positive HuH-7 cells expressed high levels of GPD2 (glycerol-3-phosphate dehydrogenase or mGPDH) and showed high G3P-ATPase activity. Next, to elucidate the relationship between CD133 and GPD2, we inhibited downstream factors of CD133 and found that a p38 inhibitor decreased the expression of GPD2 and decreased the G3P-ATPase activity. Furthermore, GPD2-knockdown (GPD2-KD) cells exhibited strong reduction of the G3P-ATPase activity and reduction of lactic acid secretion. Finally, we validated the effect of GPD2-KD on tumorigenicity. GPD2-KD cells were found to show decreased anchorage-independent cell proliferation, suggesting the linkage of G3P-ATPase activity to the tumorigenicity of the CD133-positive HuH-7 cells. Inhibition of G3P-ATPase disrupts the homeostasis of energy metabolism and blocks cancer development and progression. Our results suggest inhibitors, targeting GPD2 may be potential new anticancer agents.

Background: CD133 is a commonly used cancer stem cell (CSC) marker in breast cancer. However, the association between CD133 expression, with clinicopathological features and prognosis in breast cancer, is poorly understood in the Indian subcontinent. This study was designed to explore the expression of CD 133 in breast carcinoma and to know its association between CD133 and clinicopathological characteristics. Methods: A total of fifty seven cases were included in the study. All the clinicopathological parameters were collected from Department of Pathology archives. Slides, blocks, clinical information, tumor size and axillary lymph node status were obtained from medical records and the pathology reports. Immunohistochemistry was done using CD 133 antibodies. Both Cytoplasmic and membranous staining was taken a positive. Scoring was done based on percentage of positive cells and intensity of staining. MS Excel, SPSS version 22 (IBM SPSS Statistics, Somers NY, USA) was used to analyze data.p value < 0.05 was considered as statistically significant. Results: Statistically significant association between the CD 133 expression and nodal metastasis, tumor stage and Nottingham prognostic index was analysed. There was no statistical correlation between CD 133 expression age, tumor grade and tumor size. The disease free survival showed the mean disease free survival of CD 133 positivity cases was 16months. And the patients who were negative for CD 133 expression had mean survival of 30 months. By the Kaplan Mayer graph it was evident that the more the CD 133 expression the lesser was the disease free survival of the patients. Conclusion: CD 133 expression was seen in 77.08% cases and was associated with tumor stage, lymph node metastasis, poor Nottingham prognostic index and worse disease free survival. An increasing trend of association was seen between CD 133 expression and Age, Tumor Size and Tumor grade.

The cellular resistance of tumors is a major obstacle for successful tumor therapy. Cluster of differentiation (CD)133 plays an important role in the regulation of drug resistance in gastric and colon cancers. However, its effect on chemotherapeutic sensitivity in adenoid cystic carcinoma (ACC) has not been fully explored. The present study discussed the specific role of CD133 in ACC drug-resistant sensitive cells. KOA-1 cells were treated with 5-fluorouracil (5-FU) and pingyangmycin (PYM) to form drug-resistant cell lines. A Cell Counting Kit-8 assay was used to detect the cell survival rate. Cell invasion was measured using a Transwell assay. The expression levels of CD133 were detected by reverse transcription-quantitative (RT-q) PCR. The expression levels of drug-resistant mRNAs and proteins were detected by RT-qPCR and immunofluorescence analyses, respectively. The CD133 were inhibited by small interfering RNA technology. The survival rate and invasive ability of KOA-1 cells were increased following the induction of drug resistance. The expression levels of CD133, multidrug resistance protein (MDR)1 and multidrug resistance-associated protein (MRP)1 were significantly increased in drug-resistant cell lines. Knockdown of CD133 expression in the resistant cell lines, KOA-1/5-FU and KOA-1/PYM, decreased the survival rate and invasive ability. The expression levels of MDR1 and MRP1 were also significantly decreased. Knockdown of CD133 expression in ACC drug-resistant cells could inhibit the viability and invasion of tumors and enhance the sensitivity of drug-resistant cells to chemotherapeutic drugs.

Retinitis pigmentosa (RP) and macular dystrophy (MD) are characterized by gradual photoreceptor death in the retina and are often associated with genetic mutations, including those in the prominin-1 (Prom1) gene. Prom1-knockout (KO) mice recapitulate key features of these diseases including light-dependent retinal degeneration and constriction of retinal blood vessels. The mechanisms underlying such degeneration have remained unclear, however. We here analysed early events associated with retinal degeneration in Prom1-KO mice. We found that photoreceptor cell death and glial cell activation occur between 2 and 3 weeks after birth. Whereas gene expression was not affected at 2 weeks, the expression of several genes was altered at 3 weeks in the Prom1-KO retina, with the expression of that for endothelin-2 (Edn2) being markedly upregulated. Expression of Edn2 was also induced by light stimulation in Prom1-KO mice reared in the dark. Treatment with endothelin receptor antagonists attenuated photoreceptor cell death, gliosis and retinal vessel stenosis in Prom1-KO mice. Our findings thus reveal early manifestations of retinal degeneration in a model of RP/MD and suggest potential therapeutic agents for these diseases. This article has an associated First Person interview with the first author of the paper., Summary: The early manifestations of an inherited retinal disease was investigated by means of a transcriptome analysis, and a possible drug-based therapeutic approach (endothelin receptor antagonists) is suggested.

Background and Aims Genomic alterations that encourage stem cell activity and hinder proper maturation are central to the development of colorectal cancer (CRC). Key molecular mediators that promote these malignant properties require further elucidation to galvanize translational advances. We therefore aimed to characterize a key factor that blocks intestinal differentiation, define its transcriptional and epigenetic program, and provide preclinical evidence for therapeutic targeting in CRC. Methods Intestinal tissue from transgenic mice and patients were analyzed by means of histopathology and immunostaining. Human CRC cells and neoplastic murine organoids were genetically manipulated for functional studies. Gene expression profiling was obtained through RNA sequencing. Histone modifications and transcription factor binding were determined with the use of chromatin immunoprecipitation sequencing. Results We demonstrate that SRY-box transcription factor 9 (SOX9) promotes CRC by activating a stem cell–like program that hinders intestinal differentiation. Intestinal adenomas and colorectal adenocarcinomas from mouse models and patients demonstrate ectopic and elevated expression of SOX9. Functional experiments indicate a requirement for SOX9 in human CRC cell lines and engineered neoplastic organoids. Disrupting SOX9 activity impairs primary CRC tumor growth by inducing intestinal differentiation. By binding to genome wide enhancers, SOX9 directly activates genes associated with Paneth and stem cell activity, including PROM1. SOX9 up-regulates prominin 1 (PROM1) via a Wnt-responsive intronic enhancer. A pentaspan transmembrane protein, PROM1 uses its first intracellular domain to support stem cell signaling, at least in part through SOX9, reinforcing a PROM1-SOX9 positive feedback loop. Conclusions These studies establish SOX9 as a central regulator of an enhancer-driven stem cell–like program and carry important implications for developing therapeutics directed at overcoming differentiation defects in CRC.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive type of adult gliomas. Despite intensive therapy including surgery, radiation, and chemotherapy, invariable tumor recurrence occurs, which suggests that glioblastoma stem cells (GSCs) render these tumors persistent. Recently, the induction of GSC differentiation has emerged as an alternative method to treat GBM, and most of the current studies aim to convert GSCs to neurons by a combination of transcriptional factors. As the tumor microenvironment is typically acidic due to increased glycolysis and consequently leads to an increased production of lactic acid in tumor cells, in the present study, the role of acid-sensing ion channel 1a (ASIC1a), an acid sensor, was explored as a tumor suppressor in gliomagenesis and stemness. The bioinformatics data from The Cancer Genome Atlas revealed that ASIC1 expression levels in GBM tumor tissues were lower than those in normal brain, and glioma patients with high ASIC1 expression had longer survival than those with low ASIC1 expression. Our immunohistochemistry data from tissue microarray revealed that ASIC1a expression was negatively associated with glioma grading. Functional studies revealed that the downregulation of ASIC1a promoted glioma cell proliferation and invasion, while upregulation of ASIC1a inhibited their proliferation and invasion. Furthermore, ASIC1a suppressed growth and proliferation of glioma cells through G1/S arrest and apoptosis induction. Mechanistically, ASIC1a negatively modulated glioma stemness via inhibition of the Notch signaling pathway and GSC markers CD133 and aldehyde dehydrogenase 1. ASIC1a is a tumor suppressor in gliomagenesis and stemness and may serve as a promising prognostic biomarker and target for GBM patients.

The therapeutic induction of senescence is a potential means to treat cancer, primarily acting through the induction of a persistent growth‐arrested state in tumors. However, recent studies have indicated that therapy‐induced senescence (TIS) in tumor cells allows for the prolonged survival of a subgroup of cells in a dormant state, with the potential to re‐enter the cell cycle along with an increased stemness gene expression. Residual cells after TIS with increased cancer stem cell phenotype may have profound implications for tumor aggressiveness and disease recurrence. Herein, we investigated senescence‐associated stemness in EpCAM+/CD133+ liver cancer stem cell and EpCAM−/CD133− nonstem cell populations in HuH7 cell line. We demonstrated that treatment with doxorubicin induces senescence in both cell populations, accompanied by a significant increase in the expression of reprogramming genes SOX2, KLF4, and c‐MYC as well as liver stemness‐related genes EpCAM, CK19, and ANXA3 and the multidrug resistance‐related gene ABCG2. Moreover, doxorubicin treatment significantly increased EpCAM + population in nonstem cells indicating senescence‐associated reprogramming of nonstem cell population. Also, Wnt/β‐catenin target genes were increased in these cells, while inhibition of this signaling pathway decreased stem cell gene expression. Importantly, Dox‐treated EpCAM−/CD133− nonstem cells had increased in vivo tumor‐forming ability. In addition, when SASP‐CM from Dox‐treated cells were applied onto hİPSC‐derived hepatocytes, senescence was induced in hepatocytes along with an increased expression of TGF‐β, KLF4, and AXIN2. Importantly, SASP‐CM was not able to induce senescence in Hep3B‐TR cells, a derivative line rendered resistant to TGF‐β signaling. Furthermore, ELISA experiments revealed that the SASP‐CM of Dox‐treated cells contain inflammatory cytokines IL8 and IP10. In summary, our findings further emphasize the importance of carefully dissecting the beneficial and detrimental aspects of prosenescence therapy in HCC and support the potential use of senolytic drugs in HCC treatment in order to eliminate adverse effects of TIS., In this study, we showed that therapy‐induced senescence promotes tumorigenicity and stemness in liver cancer cells. Doxorubicin treatment induced senescence in both EpCAM+/CD133+ liver cancer stem cells and EpCAM−/CD133− nonstem cells. Doxorubicin‐treated EpCAM−/CD133− cells had higher expression of stemness‐related genes and displayed higher tumor‐initiating capacity than their non‐senescent counterparts. Notably, conditioned media from senescent doxorubicin‐treated EpCAM−/CD133− nonstem cells induced senescence and stemness in healthy hepatocytes.

To identify drivers of sarcoma cancer stem-like cells (CSCs), we compared gene expression using RNA sequencing between HT1080 fibrosarcoma and SK-LMS-1 leiomyosarcoma spheroids (which are enriched for CSCs) compared with the parent populations. The most overexpressed survival signaling-related gene in spheroids was phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of PI3K, which functions in tumorigenesis and metastasis. In a human sarcoma microarray, PIK3R3 was also overexpressed by 4.1-fold compared with normal tissues. PIK3R3 inhibition using shRNA in the HT1080, SK-LMS-1, and DDLS8817 dedifferentiated liposarcoma in spheroids and in CD133+ cells (a CSC marker) reduced expression of CD133 and the stem cell factor Nanog and blocked spheroid formation by 61–71%. Mechanistic studies showed that in spheroid cells, PIK3R3 activated AKT and ERK signaling. Inhibition of PIK3R3, AKT, or ERK using shRNA or inhibitors decreased expression of Nanog, spheroid formation by 68–73%, and anchorage-independent growth by 76–91%. PIK3R3 or ERK1/2 inhibition similarly blocked sarcoma spheroid cell migration, invasion, secretion of MMP-2, xenograft invasion into adjacent normal tissue, and chemotherapy resistance. Together, these results show that signaling through the PIK3R3/ERK/Nanog axis promotes sarcoma CSC phenotypes such as migration, invasion, and chemotherapy resistance, and identify PIK3R3 as a potential therapeutic target in sarcoma.

Dysregulated hematological and neurological expressed 1-like (HN1L) has been implicated in carcinogenesis of difference cancers, including hepatocellular carcinoma and breast cancer. However, the role of HN1L in the progression of prostate cancer (PCA) remains unknown. Therefore, we aimed to investigate the role of HN1L in stemness and progression of PCA. The expression of HN1L in PCA tissues and cells was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blot analysis, and/or immunohistochemistry (IHC). CD133+ cells were sorted from PCA cells using magnetic fluorescence cell sorting technology and were considered as cancer stem cells (CSCs). Sphere formation assays, transwell assays, and animal experiments were conducted to assess cell stemness, migration, invasion, and in vivo tumorigenesis, respectively. The results showed that HN1L expression was higher in PCA tissues and cells as compared with normal tissues and cells, as well as in CD133+ cells as compared with CD133- cells. HN1L knockdown significantly decreased the expression levels of CSC markers including OCT4 (POU class 5 homeobox 1), CD44, and SRY-box transcription factor 2, inhibited cell migration, invasion, and tumorigenesis and decreased the number of tumor spheroids and CD133+ cell population. Furthermore, we found that HN1L could bind to forkhead box P2 (FOXP2) and positively regulated transforming growth factor-β (TGF-β) expression via upregulation of FOXP2. In addition, the overexpression of TGF-β in HN1L-knockdown PCA cells increased the number of tumor spheroids and CD133+ cell population, as well as enhanced cell migration and invasion. Collectively, this study demonstrates that HN1L promotes stem cell-like properties and cancer progression by targeting FOXP2 through TGF-β signaling pathway in PCA.

BACKGROUND: CD133 (prominin-1) is the most commonly used molecular marker of the cancer stem cells (CSCs) that maintain tumor progression and recurrence in colorectal cancer. However, the proteome of CSCs directly isolated from colorectal tumors based on CD133 expression has never been investigated. OBJECTIVE: To reveal biomarkers of CD133-positive colorectal CSCs. METHODS: Thirty colorectal tumor samples were collected from patients undergoing bowel resection. CD133-positive and CD133-negative cells were isolated by FACS. Comparative proteomic profiling was performed by LC-MS/MS analysis combined with label-free quantification. Verification of differentially expressed proteins was performed by flow cytometry or ELISA. CD133-knockout Caco-2 and HT-29 cell lines were generated using CRISPR-Cas9 gene editing. RESULTS: LC-MS/MS analysis identified 29 proteins with at least 2.5-fold higher expression in CD133-positive cells versus CD133-negative cells. Flow cytometry confirmed CEACAM5 overexpression in CD133-positive cells in all clinical samples analyzed. S100A8, S100A9, and DEFA1 were differentially expressed in only a proportion of the samples. CD133 knockout in the colon cancer cell lines Caco-2 and HT-29 did not affect the median level of CEACAM5 expression, but led to higher variance of the percentage of CEACAM5-positive cells. CONCLUSIONS: High CEACAM5 expression in colorectal cancer cells is firmly associated with the CD133-positive colorectal CSC phenotype, but it is unlikely that CD133 directly regulates CEACAM5 expression.

Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133high and CD133low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths, but its molecular mechanisms are not yet well characterized. Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, including that of HCC. However, the role of homeobox A11 antisense (HOXA11-AS) in determining HCC stem cell characteristics remains to be explained; hence, this study aimed to investigate the effects of HOXA11-AS on HCC stem cell characteristics. Initially, the expression patterns of HOXA11-AS and HOXA11 in HCC tissues, cells, and stem cells were determined. HCC stem cells, successfully sorted from Hep3B and Huh7 cells, were transfected with short hairpin or overexpression plasmids for HOXA11-AS or HOXA11 overexpression and depletion, with an aim to study the influences of these mediators on the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo. Additionally, the potential relationship and the regulatory mechanisms that link HOXA11-AS, HOXA11, and the Wnt signaling pathway were explored through treatment with Dickkopf-1 (a Wnt signaling pathway inhibitor). HCC stem cells showed high expression of HOXA11-AS and low expression of HOXA11. Both HOXA11-AS silencing and HOXA11 overexpression suppressed the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo, as evidenced by the decreased expression of cancer stem cell surface markers (CD133 and CD44) and stemness-related transcription factors (Nanog, Sox2, and Oct4). Moreover, silencing HOXA11-AS inactivated the Wnt signaling pathway by decreasing the methylation level of the HOXA11 promoter, thereby inhibiting HCC stem cell characteristics. Collectively, this study suggested that HOXA11-AS silencing exerts an antitumor effect, suppressing HCC development via Wnt signaling pathway inactivation by decreasing the methylation level of the HOXA11 promoter., Liver cancer: An RNA molecule that triggers tumor growth A long RNA molecule promotes the growth of liver cancer cells through its inhibitory effects on gene regulation. The HOXA11 gene controls cell proliferation and tissue development, and several studies have suggested that HOXA11-AS, an RNA that regulates this gene, may play a role in certain cancers. Researchers led by Min Guo at Hainan General Hospital in Haikou, China, have now obtained evidence linking HOXA11-AS to the growth of hepatocellular carcinoma cells. After determining that this RNA is consistently highly expressed in such cells, the authors demonstrated that it can stimulate cellular proliferation and invasive behavior through its suppressive effects on HOXA11 and other genes. This inhibition results from HOXA11-AS-induced chemical modification of these DNA sequences. The authors hypothesize that this same mechanism could also contribute to growth of other tumor subtypes.

Stem cell-like-cancer cells are key drivers of tumor growth, metastasis, and relapse of cancer following remission. Prostate stem cell-like cancer cells isolated from human prostate cancer (PC) biopsies express CD44+/α2β1 hi/CD133+ cell surface markers and can self-renew in vitro. Expression of calcitonin (CT) and its receptor (CTR) is frequently elevated in PCs and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We investigated whether CT-CTR autocrine axis induces stem cell-like phenotype in two PC cell lines. CT-CTR axis in these cell lines was activated by enforced expression of CTR. The cells were then examined for the changes in the expression of CD44 and CD133, collagen adherence, tumorigenic, metastatic and repopulating characteristics. The activation of CT-CTR axis led to a large increase in adherence to collagen and a remarkable increase of CD44 and CD133 in PC-3 and LNCaP cells. This was accompanied by a strong increase in tumorigenic, metastatic and repopulation properties of PC cells. However, the mutation of CTR-C PDZ-binding site in CTR almost abolished CTR-mediated increases in stem cell-like characteristics of PC cells. These results support an important role for CT-CTR axis in the progression of PC from localized cancer to an aggressive form, and a majority of proinvasive CTR actions may be mediated through its interaction with its partner protein at the PDZ-binding site. These results suggest that CT/CTR can serve as a valuable target to prevent the generation of stem-like PC cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Anoikis resistance is an essential property of cancer cells that allow the extra-cellular matrix-detached cells to survive in a suspended state in body fluid in order to metastasize and invade to distant organs. It is known that integrins play an important role in anoikis resistance, but detailed mechanisms are not well understood. Here we report that highly metastatic colon cancer cells showed a higher degree of anoikis resistance than the normal intestinal epithelial cells. These anoikis-resistant cancer cells express high-levels of integrin-α2, β1, and activated EGFR in the anchorage-independent state than the anchorage-dependent state. In contrast, normal intestinal epithelial cells failed to elevate these proteins. Interestingly, a higher co-association of EGFR with integrin-α2β1/-α5β1 was observed on the surface of anoikis-resistant cells. Thus, in the absence of extra-cellular matrix, integrins in association with EGFR activates downstream effectors ERK and AKT and suppress Caspase-3 activation to induce anoikis resistance as was confirmed from the gene-ablation and pharmacological inhibitor studies. Interestingly, these anoikis-resistant cancer cells express high-level of cancer stem cell signatures (CD24, CD44, CD133, EpCAM) and pluripotent stem cell markers (OCT-4, SOX-2, Nanog) as well as drug-resistant pumps (ABCG2, MDR1, MRP1). Altogether, our findings unravel the interplay between integrin-α2β1/-α5β1 and EGFR in anoikis resistance and suggest that the resistant cells are cancer initiating or cancer stem cells, which may serve as a promising target to combat metastasis of cancer.

In the United States, Hepatocellular Carcinoma (HCC) incidence has tripled over the past two decades. The disease has disproportionately affected minority and disadvantaged populations. The purpose of this study was to examine the expression of SATB2 gene in HCC cells derived from African Americans (AA) and Caucasian Americans (CA) and assess its oncogenic potential by measuring cell viability, spheroid formation, epithelial‐mesenchymal transition (EMT), stem cell markers and pluripotency maintaining factors in cancer stem cells (CSCs). We compared the expression of SATB2 in human primary hepatocytes, HCC cells derived from AA and CA, and HCC CSCs. Hepatocellular carcinoma cells derived from AA expressed the higher level of SATB2 than those from CA. By comparison, normal human hepatocytes did not express SATB2. Higher expression of SATB2 in HCC cells from AA was associated with greater growth rate, cell viability, colony formation and EMT characteristics than those from CA. Knockout of SATB2 in CSCs by Crispr/Cas9 technique significantly inhibited the expression of SATB2 gene, stem cell markers (CD24, CD44 and CD133), pluripotency maintaining factors (c‐Myc, KLF4, SOX2 and OCT4), and EMT compared with non‐targeting control group. The expression of SATB2 was negatively correlated with miR34a. SATB2 rescued the miR‐34a‐mediated inhibition of CSC's viability. These data suggest that SATB2 is an oncogenic factor, and its higher expression may explain the disparity in HCC outcomes among AA.

Restoring β-cell mass by the transplantation of pancreatic islets is an effective diabetes treatment, but it is limited by the shortage of donor organs. CD133-expressing pancreatic ductal epithelial cells (PDECs) have the ability to generate insulin-producing cells. The expansion of these cells is dependent on extrinsic niche factors, but few of those signals have been identified. In this study, CD133-expressing PDECs were purified by sorting from adult wild-type C57BL/6 mice and TGFβRII

Background information Metastatic disease is caused by the ability of cancer cells to reach distant organs and form secondary lesions at new locations. Dissemination of cancer cells depends on their migration plasticity - an ability to switch between motility modes driven by distinct molecular machineries. One of such switches is mesenchymal-to-amoeboid transition. Although mesenchymal migration of individual cells requires Arp2/3-dependent actin polymerisation, amoeboid migration is characterised by a high level of actomyosin contractility and often involves the formation of membrane blebs. The acquisition of amoeboid motility by mesenchymal cells is often associated with enhanced metastasis. Results We studied the ability of mesenchymal HT1080 fibrosarcoma cells to switch to amoeboid motility. We induced the transition from lamellipodium-rich to blebbing phenotype either by down-regulating the Arp2/3 complex, pharmacologically or by RNAi, or by decreasing substrate adhesiveness. Each of these treatments induced blebbing in a subset of fibrosarcoma cells, but not in normal subcutaneous fibroblasts. A significant fraction of HT1080 cells that switched to blebbing behaviour exhibited stem cell-like features, such as expression of the stem cell marker CD133, an increased efflux of Hoechst-33342 and positive staining for Oct4, Sox2 and Nanog. Furthermore, the isolated CD133+ cells demonstrated an increased ability to switch to bleb-rich amoeboid phenotype both under inhibitor's treatment and in 3D collagen gels. Conclusions Together, our data show a significant correlation between the increased ability of cells to switch between migration modes and their stem-like features, suggesting that migration plasticity is an additional property of stem-like population of fibrosarcoma cells. This combination of features could facilitate both dissemination of these cells to distant locations, and their establishment self-renewal in a new microenvironment, as required for metastasis formation. Significance These data suggest that migration plasticity is a new feature of cancer stem-like cells that can significantly facilitate their dissemination to a secondary location by allowing them to adapt quickly to challenging microenvironments. Moreover, it complements their resistance to apoptosis and self-renewal potential, thus enabling them not only to disseminate efficiently, but also to survive and colonise new niches.

Physical cues are vital in determining cellular fate in cancer. In vitro 3D culture do not replicate forces present in vivo. These forces including tumor interstitial fluid pressure and matrix stiffness behave as switches in differentiation and metastasis, which are intricate features of cancer stem cells (CSCs). Gravity determines the effect of these physical factors on cell fate and functions as evident from microgravity experiments on space and ground simulations. Here, we described the role of simulation of microgravity (SMG) using rotary cell culture system (RCCS) in increasing stemness in human colorectal cancer cell HCT116. We observed distinct features of cancer stem cells including CD133/CD44 dual positive cells and migration in SMG which was not altered by autophagy induction or inhibition. 3D and SMG increased autophagy, but the flux was staggered under SMG. Increased unique giant cancer cells housing complete nuclear localization of YAP were observed in SMG. This study highlights the role of microgravity in regulating stemness in CSC and importance of physical factors in determining the same.

Triple-negative breast cancer (TNBC) is an aggressive disease with a short median time from relapse to death. The increased aggressiveness, drug resistance, disease relapse, and metastasis are associated with the presence of stem cells within tumors. Several stem cell markers, such as CD24, CD44, CD133, ALDH1, and ABCG2, have been reported, but their roles in breast cancer tumorigenesis remain unclear. Herein, we apply RNA nanotechnology to deliver anti-microRNA (miRNA) for TNBC therapy. The thermodynamically and chemically stable three-way junction (3WJ) motif was utilized as the scaffold to carry an RNA aptamer binding to CD133 receptor and a locked nuclei acid (LNA) sequence for miRNA21 inhibition. Binding assays revealed the specific uptake of the nanoparticles to breast cancer stem cells (BCSCs) and TNBC cells. Functional assays showed that cancer cell migration was reduced, miR21 expression was inhibited, and downstream tumor suppressor PTEN and PDCD4 expressions were upregulated. In vitro and in vivo studies revealed that these therapeutic RNA nanoparticles did not induce cytokine secretion. Systemic injection of these RNA nanoparticles in animal trial demonstrated high specificity in TNBC tumor targeting and high efficacy for tumor growth inhibition. These results revealed the clinical translation potential of these RNA nanoparticles for TNBC therapy.

Transplanted stromal cells have demonstrated considerable promise as therapeutic agents in diverse disease settings. Paracrine signaling can be an important mediator of these therapeutic effects at the sites of acute or persistent injury and inflammation. As many stromal cell types, including bone marrow-derived stromal cells (BMSCs), display tissue-specific responses, there is a need to explore their secretory dynamics in the context of tissue and injury type. Paracrine signals are not static, and could encode contextual dynamics in the kinetic changes of the concentrations of the secreted ligands. However, precise measurement of dynamic and context-specific cellular secretory signatures, particularly in adherent cells, remains challenging. Here, by creating an experimental and computational analysis platform, we reconstructed dynamic secretory signatures of cells based on a very limited number of time points. By using this approach, we demonstrate that the secretory signatures of CD133-positive BMSCs are uniquely defined by distinct biological contexts, including signals from injured cardiac cells undergoing oxidative stress, characteristic of cardiac infarction. Furthermore, we show that the mixture of recombinant factors reproducing the dynamics of BMSC-generated secretion can mediate a highly effective rescue of cells injured by oxidative stress and an improved cardiac output. These results support the importance of the dynamic multifactorial paracrine signals in mediating remedial effects of stromal stem cells, and pave the way for stem cell-inspired cell-free treatments of cardiac and other injuries.

Retinoblastoma (RB) is a primary intraocular malignancy in childhood, and may develop relapse and metastatic disease. This study was to identify the stem-cell properties of primary retinoblastoma cells critical to tumorigenesis and metastasis. Primary cells were isolated from fresh human RB tissues after enucleation, and cultured in serum-free or serum-enriched conditions, with two RB cell-lines Weri-RB1 and Y79 for comparison. Proliferation of primary RB cells were well-maintained in serum-free condition of DMEM/F12 medium, and formed stem-cell like spheroids. The immaturity of cultured primary RB cells was demonstrated by tendency of highly expressed stem-cell markers (CD133, Nestin and OCT4) and suppressed mature retinal-cell markers (GFAP, MAP2 and Recoverin). CD133, a neural stem-cell marker being exclusively studied in RB, was found positive in small patches of cells in archival human RB by immunohistochemistry. Meanwhile, at initial isolation, insignificant CD133+ cells were detected by flow-cytometry, and substantial increase of positivity was observed after several days cultivated in serum-free condition. Cultured primary RB cells were engrafted in subretinal region of BALB/c nude mice for assessment of tumorigenicity. Strong tumorigenic activity and extensive progression of the xenograft retinoblastoma was induced by primary cells as compared with the two cell-lines. Again, immunohistochemistry confirmed that the stem-cell markers were emphasized in the xenograft tumor in mice. Our findings demonstrated that in comparison to the well-established RB cell-lines, cultured primary RB cells possess stem-cell like properties with highly expressed stem-cell markers, self-regenerative growth in culture, and strong in vivo oncogenic potentiality.

Accumulation of lactate in tumor has been linked to poor prognosis of oral squamous cell carcinoma (OSCC), but the underlying mechanism remained largely uncertain. Previous studies have suggested that presence of cancer stem cells (CSCs) closely correlated with cellular malignancy of OSCC. Here, using 3D organoid culture model, we investigated whether lactate promoted CSCs phenotype in primary OSCC cells. We generated organoids using fresh OSCC specimens and verified that organoids recapitulated histopathology and cellular heterogeneity of parental tumor. Organoids were then transfected with a Wnt reporter to visualize Wnt activity. The sphere forming assay demonstrated that high Wnt activity functionally designated CSCs population in OSCC cells. Further investigations indicated that lactate treatment promoted Wnt activity and increased the expression of CSCs (i.e. CD133+ cells) in organoids. Moreover, silencing monocarboxylate transporter 1 (MCT1), the prominent path for lactate uptake in human tumor with siRNA significantly impaired organoid forming capacity of OSCC cells. Together, our study demonstrated that lactate can promote CSCs phenotype of OSCC, and MCT1 may be a therapeutic target against OSCC growth.

Background: Cancer stem cells (CSCs) with a self-renewal ability in tumor cells population, execute a pivotal function in tumorigenesis, retrogression, and metastasis of malignant cancers such as anaplastic thyroid carcinoma (ATC). Materials and Methods: In this study, we isolated CSCs subpopulation with CD133 surface marker from three ATC cell lines by magnetic cell sorting assay. After confirming the segregation by the flow cytometry method, BRAF and sodium-iodide symporter (NIS) genes were investigated in them before and after incubation with BRAF inhibitor. Also, we evaluated the NIS protein expression and localization. Results: Established upon q-RT PCR data, when compared to human normal thyrocytes, the BRAFV600E gene was over-expressed in CD133pos cells (>1705.99 ± 55.55 fold, Mean ± SEM, n=3, P- value