Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

Cell softness regulates tumorigenicity and stemness of cancer cells.

Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells (< 400 Pa) but not the stiff ones (> 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.

RASSF1C oncogene elicits amoeboid invasion, cancer stemness, and extracellular vesicle release via a SRC/Rho axis.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

CD44 and CD133 protein expression might serve as a prognostic factor for early occurrence castration-resistant prostate cancer.

BACKGROUND: The occurrence of castration-resistant prostate cancer (CRPC) varies in patients with advanced prostate cancer (PCa) undergoing androgen deprivation therapy (ADT). The rate of occurrence of CRPC may be related to the presence of prostate cancer stem cells (CSC). Thus, this study aims to evaluate the presence of CSC markers (CD44 and CD133) in histopathology tissue at the time of diagnosis and their correlation with the occurrence of CRPC in patients with advanced PCa within 2 years of ADT. METHOD: A retrospective case-control study was conducted to evaluate the incidence of CRPC within 2 years. The inclusion criteria were patients with PCa who had received treatment with ADT and a first-generation anti-androgen (AA) for 2 years. We classified patients based on whether they developed CRPC within 2 years (CRPC) of the therapy or did not experience CRPC within 2 years (non-CRPC) of the therapy. We performed immunohistochemical (IHC) staining for CD44 and CD133 on the prostate biopsy tissue samples. RESULTS: Data were collected from records spanning 2011-2019. We analyzed a total of 65 samples, including 22 patients with CRPC and 43 patients with non-CRPC who had received treatment with LHRH agonists and AA for up to 2 years. Our findings showed a significant H-score difference in CD44 protein expression between CRPC prostate adenocarcinoma samples 869 (200-1329) and non-CRPC 524 (154-1166) (p = 0.033). There was no significant difference in CD133 protein expression between the two groups (p = 0.554). However, there was a significant difference in the nonoccurrence of CRPC between the high expressions of both CD44 and CD133 groups with other expressions of CD44/CD133 groups (25% vs. 75%; p = 0.011; odds ratio = 4.29; 95% confidence interval [1.34, 13.76]). CONCLUSION: This study found a low expression of at least one CD44/CD133 protein in the patients without early occurrence of CRPC. This result might suggest that CD44/CD133 may function as a potential prognostic marker for PCa, especially in a low expression, to identify patients who have a better prognosis regarding the occurrence of early CRPC.

CD57 defines a novel cancer stem cell that drive invasion of diffuse pediatric-type high grade gliomas.

BACKGROUND: Diffuse invasion remains a primary cause of treatment failure in pediatric high-grade glioma (pHGG). Identifying cellular driver(s) of pHGG invasion is needed for anti-invasion therapies. METHODS: Ten highly invasive patient-derived orthotopic xenograft (PDOX) models of pHGG were subjected to isolation of matching pairs of invasive (HGGINV) and tumor core (HGGTC) cells. RESULTS: pHGGINV cells were intrinsically more invasive than their matching pHGGTC cells. CSC profiling revealed co-positivity of CD133 and CD57 and identified CD57+CD133- cells as the most abundant CSCs in the invasive front. In addition to discovering a new order of self-renewal capacities, i.e., CD57+CD133- > CD57+CD133+ > CD57-CD133+ > CD57-CD133- cells, we showed that CSC hierarchy was impacted by their spatial locations, and the highest self-renewal capacities were found in CD57+CD133- cells in the HGGINV front (HGGINV/CD57+CD133- cells) mediated by NANOG and SHH over-expression. Direct implantation of CD57+ (CD57+/CD133- and CD57+/CD133+) cells into mouse brains reconstituted diffusely invasion, while depleting CD57+ cells (i.e., CD57-CD133+) abrogated pHGG invasion. CONCLUSION: We revealed significantly increased invasive capacities in HGGINV cells, confirmed CD57 as a novel glioma stem cell marker, identified CD57+CD133- and CD57+CD133+ cells as a new cellular driver of pHGG invasion and suggested a new dual-mode hierarchy of HGG stem cells.

Glutamine deprivation in glioblastoma stem cells triggers autophagic SIRT3 degradation to epigenetically restrict CD133 expression and stemness.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor, and glioblastoma stem cells (GSCs) are the primary cause of GBM heterogeneity, invasiveness, and resistance to therapy. Sirtuin 3 (SIRT3) is mainly localized in the mitochondrial matrix and plays an important role in maintaining GSC stemness through cooperative interaction with the chaperone protein tumor necrosis factor receptor-associated protein 1 (TRAP1) to modulate mitochondrial respiration and oxidative stress. The present study aimed to further elucidate the specific mechanisms by which SIRT3 influences GSC stemness, including whether SIRT3 serves as an autophagy substrate and the mechanism of SIRT3 degradation. We first found that SIRT3 is enriched in CD133+ GSCs. Further experiments revealed that in addition to promoting mitochondrial respiration and reducing oxidative stress, SIRT3 maintains GSC stemness by epigenetically regulating CD133 expression via succinate. More importantly, we found that SIRT3 is degraded through the autophagy-lysosome pathway during GSC differentiation into GBM bulk tumor cells. GSCs are highly dependent on glutamine for survival, and in these cells, we found that glutamine deprivation triggers autophagic SIRT3 degradation to restrict CD133 expression, thereby disrupting the stemness of GSCs. Together our results reveal a novel mechanism by which SIRT3 regulates GSC stemness. We propose that glutamine restriction to trigger autophagic SIRT3 degradation offers a strategy to eliminate GSCs, which combined with other treatment methods may overcome GBM resistance to therapy as well as relapse.

GLP-1RA therapy increases circulating vascular regenerative cell content in people living with type 2 diabetes.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) and sodium-glucose cotransporter-2 (SGLT2) inhibitors are guideline-recommended therapies for the management of type 2 diabetes (T2D), atherosclerotic cardiovascular disease, heart failure, and chronic kidney disease. We previously observed in people living with T2D and coronary artery disease that circulating vascular regenerative (VR) progenitor cell content increased following 6-mo use of the SGLT2 inhibitor empagliflozin. In this post hoc subanalysis of the ORIGINS-RCE CardioLink-13 study (ClinicalTrials.gov Identifier NCT05253521), we analyzed the circulating VR progenitor cell content of 92 individuals living with T2D, among whom 20 were on a GLP-1RA, 42 were on an SGLT2 inhibitor but not a GLP-1RA, and 30 were on neither of these vascular protective therapies. In the GLP-1RA group, the mean absolute count of circulating VR progenitor cells defined by high aldehyde dehydrogenase (ALDH) activity (ALDHhiSSClow) and VR progenitor cells further characterized by surface expression of the proangiogenic marker CD133 (ALDHhiSSClowCD133+) was higher than the group receiving neither a GLP-1RA nor an SGLT2 inhibitor (P = 0.02) and comparable with that in the SGLT2 inhibitor group (P = 0.25). The absolute count of proinflammatory, granulocyte-restricted precursor cells (ALDHhiSSChi) was significantly lower in the GLP-1RA group compared with the group on neither therapy (P = 0.031). Augmented vessel repair initiated by VR cells with previously documented proangiogenic activity, alongside a reduction in systemic, granulocyte precursor-driven inflammation, may represent novel mechanisms responsible for the cardiovascular-metabolic benefits of GLP-1RA therapy. Prospective, randomized clinical trials are now warranted to establish the value of recovering circulating VR progenitor cell content with blood vessel regenerative functions.NEW & NOTEWORTHY In this post hoc subanalysis of 92 individuals living with T2D and at high cardiovascular risk, the authors summarize the differences in circulating vascular regenerative (VR) progenitor cell content between those on GLP-1RA therapy, on SGLT2 inhibitor without GLP-1RA therapy, and on neither therapy. Those on GLP-1RA therapy demonstrated greater circulating VR progenitor cell content and reduced proinflammatory granulocyte precursor content. These results offer novel mechanistic insights into the cardiometabolic benefits associated with GLP-1RA therapy.

CD133 expression is associated with less DNA repair, better response to chemotherapy and survival in ER-positive/HER2-negative breast cancer.

PURPOSE: CD133, a cancer stem cells (CSC) marker, has been reported to be associated with treatment resistance and worse survival in triple-negative breast cancer (BC). However, the clinical relevance of CD133 expression in ER-positive/HER2-negative (ER + /HER2-) BC, the most abundant subtype, remains unknown. METHODS: The BC cohorts from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n = 1904) and The Cancer Genome Atlas (TCGA, n = 1065) were used to obtain biological variables and gene expression data. RESULTS: Epithelial cells were the exclusive source of CD133 gene expression in a bulk BC. CD133-high ER + /HER2- BC was associated with CD24, NOTCH1, DLL1, and ALDH1A1 gene expressions, as well as with WNT/ß-Catenin, Hedgehog, and Notch signaling pathways, all characteristic for CSC. Consistent with a CSC phenotype, CD133-low BC was enriched with gene sets related to cell proliferation, such as G2M Checkpoint, MYC Targets V1, E2F Targets, and Ki67 gene expression. CD133-low BC was also linked with enrichment of genes related to DNA repair, such as BRCA1, E2F1, E2F4, CDK1/2. On the other hand, CD133-high tumors had proinflammatory microenvironment, higher activity of immune cells, and higher expression of genes related to inflammation and immune response. Finally, CD133-high tumors had better pathological complete response after neoadjuvant chemotherapy in GSE25066 cohort and better disease-free survival and overall survival in both TCGA and METABRIC cohorts. CONCLUSION: CD133-high ER + /HER2- BC was associated with CSC phenotype such as less cell proliferation and DNA repair, but also with enhanced inflammation, better response to neoadjuvant chemotherapy and better prognosis.

High CD133 expression in proximal tubular cells in diabetic kidney disease: good or bad?

BACKGROUND: Proximal tubular cells (PTCs) play a critical role in the progression of diabetic kidney disease (DKD). As one of important progenitor markers, CD133 was reported to indicate the regeneration of dedifferentiated PTCs in acute kidney disease. However, its role in chronic DKD is unclear. Therefore, we aimed to investigate the expression patterns and elucidate its functional significance of CD133 in DKD. METHODS: Data mining was employed to illustrate the expression and molecular function of CD133 in PTCs in human DKD. Subsequently, rat models representing various stages of DKD progression were established. The expression of CD133 was confirmed in DKD rats, as well as in human PTCs (HK-2 cells) and rat PTCs (NRK-52E cells) exposed to high glucose. The immunofluorescence and flow cytometry techniques were utilized to determine the expression patterns of CD133, utilizing proliferative and injury indicators. After overexpression or knockdown of CD133 in HK-2 cells, the cell proliferation and apoptosis were detected by EdU assay, real-time cell analysis and flow analysis. Additionally, the evaluation of epithelial, progenitor cell, and apoptotic indices was performed through western blot and quantitative RT-PCR analyses. RESULTS: The expression of CD133 was notably elevated in both human and rat PTCs in DKD, and this expression increased as DKD progressed. CD133 was found to be co-expressed with CD24, KIM-1, SOX9, and PCNA, suggesting that CD133+ cells were damaged and associated with proliferation. In terms of functionality, the knockdown of CD133 resulted in a significant reduction in proliferation and an increase in apoptosis in HK-2 cells compared to the high glucose stimulus group. Conversely, the overexpression of CD133 significantly mitigated high glucose-induced cell apoptosis, but had no impact on cellular proliferation. Furthermore, the Nephroseq database provided additional evidence to support the correlation between CD133 expression and the progression of DKD. Analysis of single-cell RNA-sequencing data revealed that CD133+ PTCs potentially play a role in the advancement of DKD through multiple mechanisms, including heat damage, cell microtubule stabilization, cell growth inhibition and tumor necrosis factor-mediated signaling pathway. CONCLUSION: Our study demonstrates that the upregulation of CD133 is linked to cellular proliferation and protects PTC from apoptosis in DKD and high glucose induced PTC injury. We propose that heightened CD133 expression may facilitate cellular self-protective responses during the initial stages of high glucose exposure. However, its sustained increase is associated with the pathological progression of DKD. In conclusion, CD133 exhibits dual roles in the advancement of DKD, necessitating further investigation.

Fatty acid oxidation is critical for the tumorigenic potential and chemoresistance of pancreatic cancer stem cells.

BACKGROUND: We have previously demonstrated the significant reliance of pancreatic Cancer Stem Cells (PaCSCs) on mitochondrial oxidative phosphorylation (OXPHOS), which enables versatile substrate utilization, including fatty acids (FAs). Notably, dysregulated lipid scavenging and aberrant FA metabolism are implicated in PDAC progression. METHODS & RESULTS: Our bioinformatics analyses revealed elevated expression of lipid metabolism-related genes in PDAC tissue samples compared to normal tissue samples, which correlated with a stemness signature. Additionally, PaCSCs exhibited heightened expression of diverse lipid metabolism genes and increased lipid droplet accumulation compared to differentiated progenies. Treatment with palmitic, oleic, and linolenic FAs notably augmented the self-renewal and chemotherapy resistance of CD133+ PaCSCs. Conversely, inhibitors of FA uptake, storage and metabolism reduced CSC populations both in vitro and in vivo. Mechanistically, inhibition of FA metabolism suppressed OXPHOS activity, inducing energy depletion and subsequent cell death in PaCSCs. Importantly, combining a FAO inhibitor and Gemcitabine treatment enhanced drug efficacy in vitro and in vivo, effectively diminishing the CSC content and functionality. CONCLUSION: Targeting FAO inhibition represents a promising therapeutic strategy against this highly tumorigenic population.

TWEAK/FN14 promotes profibrogenic pathway activation in Prominin-1-expressing hepatic progenitor cells in biliary atresia.

BACKGROUND AND AIMS: Biliary atresia (BA), a congenital cholestatic liver disease, commonly culminates in end-stage liver disease. We previously demonstrated in BA that Prominin-1 ( Prom1 )-expressing hepatic progenitor cells (HPCs) expand within regions of developing fibrosis, giving rise to cholangiocytes within biliary ductular reactions. Null mutation of Prom1 or ablation of cells expressing Prom1 significantly diminishes fibrogenesis. FN14, the receptor for TNF-like weak inducer of apoptosis (TWEAK), is expressed by HPCs. TWEAK/FN14 signaling promotes fibrosis in multiple organ systems. Therefore, we hypothesized that TWEAK/FN14 signaling mediates Prom1 -expressing HPC proliferation leading to profibrogenic ductular reactions in BA. APPROACH AND RESULTS: The experimental mouse model of BA mediated by perinatal rhesus rotavirus (RRV) infection resulted in increased co-expression of Fn14 in Prom1 -expressing HPCs within regions of ductular reactions. FN14 antagonist L524-0366 decreased ductular reactions, biliary fibrosis and periportal fibroblast activation in RRV injury. L524-0366 inhibition also demonstrated loss of downstream noncanonical NF-kB signaling expression in RRV injury. Murine HPC organoids demonstrated accelerated organoid growth and proliferation when treated with recombinant TWEAK. Increased organoid proliferation with recombinant TWEAK was lost when also treated with L524-0366. Analysis of a large publicly available RNA sequencing database of BA and normal control patients revealed significant increases in expression of PROM1 , FN14 , and genes downstream of TNF signaling and noncanonical NF-κB signaling pathways in BA infants. Infants who failed to achieve bile drainage after hepatoportoenterostomy had higher relative levels of FN14 expression. CONCLUSION: TWEAK/FN14 signaling activation in Prom1 -expressing HPCs contributes to proliferation of profibrogenic ductular reactions in BA.

BRAF mutations and the association of V600E with CD133 and CDX2 expression in a Pakistani colorectal carcinoma cohort.

BACKGROUND: Despite a high incidence of colorectal carcinoma, data regarding genetic aberrations in colorectal carcinoma (CRC) patients in Pakistan is scarce. This study aimed to determine the frequency of BRAFV600E mutations in colorectal carcinoma tissue in the Pakistani population and to associate BRAFV600E expression with CD133, a marker of colorectal stem cells, and CDX2 marker of differentiation. METHODS: Sanger Sequencing of exon 15 (426 bp) including the hotspot V600E was performed on formalin-fixed-paraffin-embedded (FFPE) CRC tissue samples of 115 patients. The samples were subjected to immunohistochemistry (IHC) to assess the expression of BRAFV600E, CDX2, and CD133. Additionally, homology modelling and docking were performed to investigate novel deletions revealed in sequencing. RESULTS: Twenty-four (20.8%) BRAF variants were identified in the coding region, with V600E mutations detected in 14 (12.2% )cases (GenBank: PP003258.1; Pop Set: 2678087296). Moreover, a wide spectrum of novel non-V600E mutations (8.6%) were identified, including deletions and missense variations. In-silico analysis revealed that due to large deletions in the coding region of three samples, the affinity of the anti-BRAF drugs (Encorafenib and Vemurafenib) for the active site decreased in comparison to the wild type. The IHC analysis showed that BRAFV600E expression was significantly associated with CD133 expression (χ2(1, n=115) = 26.351; p = < 0.001) and with CDX2 expression (χ2(1, n=115) = 14.88; p = 0.001). Multivariate analysis using binary logistic regression revealed association of BRAFV600E mutations with age (OR = 1.123; CI = 1.024-1.232; p = 0.014), gender (OR = 0.071; CI = 0.006-0.831; p = 0.035), grade (0.007; CI = 0-0.644) and CD133 expression (OR = 65.649; CI = 2.153-2001.556; p = 0.016). CONCLUSION: The present study demonstrates a notably high V600E frequency (12.2%) in comparison to global reported data, which ranges from 0.4 to 18%. This finding reflects the importance of upfront BRAF testing of the genetically distinct population of Pakistan. Previously unreported mutations identified in the sample may be of clinical significance and warrant further investigation. The concomitant high expression and significant association between CD133 and BRAFV600E represent vital actionable genes that may be targeted together to improve CRC patient management.

The Effect of SGLT2 Inhibitor Therapy on Endothelial Progenitor Cell Function in Patients With Heart Failure.

ABSTRACT: Sodium-glucose cotransporter-2 (SGLT-2) inhibitors have been shown to reduce the risk of cardiovascular mortality and hospitalizations in patients with heart failure (HF) with preserved or reduced ejection fraction (HFpEF or HFrEF). The mechanism for this benefit is not clear. Endothelial progenitor cells (EPCs) are bone marrow-derived cells able to differentiate into functional endothelial cells and participate in endothelial repair. The aim of this study was to evaluate the effect of SGLT-2 inhibitors on the level and function of EPCs in patients with HF. We enrolled 20 patients with symptomatic HF, 12 with HFrEF and 8 with HFpEF (aged 73.3 ± 10.2 years, 95% men). Blood samples were drawn at 2 time points: baseline and ≥3 months after initiation of SGLT-2 inhibitor therapy. Circulating EPC levels were evaluated by expression of vascular endothelial growth factor receptor-2 (VEGFR-2), CD34, and CD133 by flow cytometry. EPC colony forming units (CFUs) were quantified after 7 days in culture. The proportion of cells that coexpressed VEGFR-2 and CD34 or VEGFR-2 and CD133 was higher following 3 months of SGLT-2 inhibitors [0.26% (interquartile range, IQR 0.10-0.33) versus 0.55% (IQR 0.28-0.91), P = 0.002; 0.12% (IQR 0.07-0.15) versus 0.24% (IQR 0.15-0.39), P = 0.001, respectively]. EPC CFUs were also increased following SGLT-2 inhibitor treatment [23 (IQR 3.7-37.8) versus 79.4 (IQR 25.1-110.25) colonies/10 6 cells, P = 0.0039]. In patients with symptomatic HF, both HFpEF and HFrEF, treatment with SGLT-2 inhibitors is associated with an increase in the level and function of circulating EPCs. This augmentation in EPCs may be a contributing mechanism to the clinical benefit of SGLT-2 inhibitors in patients with HF.

Beta hydroxybutyrate induces lung cancer cell death, mitochondrial impairment and oxidative stress in a long term glucose-restricted condition.

BACKGROUND: Metabolic plasticity gives cancer cells the ability to shift between signaling pathways to facilitate their growth and survival. This study investigates the role of glucose deprivation in the presence and absence of beta-hydroxybutyrate (BHB) in growth, death, oxidative stress and the stemness features of lung cancer cells. METHODS AND RESULTS: A549 cells were exposed to various glucose conditions, both with and without beta-hydroxybutyrate (BHB), to evaluate their effects on apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) levels using flow cytometry, and the expression of CD133, CD44, SOX-9, and ß-Catenin through Quantitative PCR. The activity of superoxide dismutase, glutathione peroxidase, and malondialdehyde was assessed using colorimetric assays. Treatment with therapeutic doses of BHB triggered apoptosis in A549 cells, particularly in cells adapted to glucose deprivation. The elevated ROS levels, combined with reduced levels of SOD and GPx, indicate that oxidative stress contributes to the cell arrest induced by BHB. Notably, BHB treatment under glucose-restricted conditions notably decreased CD133 expression, suggesting a potential inhibition of cell survival through the downregulation of CD133 levels. Additionally, the simultaneous decrease in mitochondrial membrane potential and increase in ROS levels indicate the potential for creating oxidative stress conditions to impede tumor cell growth in such environmental settings. CONCLUSION: The induced cell death, oxidative stress and mitochondria impairment beside attenuated levels of cancer stem cell markers following BHB administration emphasize on the distinctive role of metabolic plasticity of cancer cells and propose possible therapeutic approaches to control cancer cell growth through metabolic fuels.

Pituitary tumours without distinct lineage differentiation express stem cell marker SOX2.

CONTEXT: The recent WHO 2022 Classification of pituitary tumours identified a novel group of 'plurihormonal tumours without distinct lineage differentiation (WDLD)'. By definition, these express multiple combinations of lineage commitment transcription factors, in a monomorphous population of cells. OBJECTIVES: To determine the expression of stem cell markers (SOX2, Nestin, CD133) within tumours WDLD, immature PIT-1 lineage and acidophil stem cell tumours, compared with committed cell lineage tumours. METHODS: Retrospective evaluation of surgically resected pituitary tumours from St Vincent's Hospital, Sydney. Patients were selected to cover a range of tumour types, based on transcription factor and hormone immunohistochemistry. Clinical data was collected from patient files. Radiology reports were reviewed for size and invasion. Samples were analysed by immunohistochemistry and RT-qPCR for SF-1, PIT-1, T-PIT, SOX2, Nestin and CD133. Stem cell markers were compared between tumours WDLD and those with classically "mature" types. RESULTS: On immunohistochemistry, SOX2 was positive in a higher proportion of tumours WDLD compared with those meeting WHO lineage criteria, 7/10 v 10/42 (70 v 23.4%, p = 0.005). CD133 was positive in 2/10 tumours WDLD but 0/41 meeting lineage criteria, P = 0.003. On RT-qPCR, there was no significant difference in relative expression of stem cell markers (SOX2, CD133, Nestin) between tumours with and WDLD. CONCLUSIONS: Our study is the first to biologically characterise pituitary tumours WDLD. We demonstrate that these tumours exhibit a higher expression of the stem cell marker SOX2 compared with other lineage-differentiated tumours, suggesting possible involvement of stem cells in their development.

Emerging roles of prominin-1 (CD133) in the dynamics of plasma membrane architecture and cell signaling pathways in health and disease.

Prominin-1 (CD133) is a cholesterol-binding membrane glycoprotein selectively associated with highly curved and prominent membrane structures. It is widely recognized as an antigenic marker of stem cells and cancer stem cells and is frequently used to isolate them from biological and clinical samples. Recent progress in understanding various aspects of CD133 biology in different cell types has revealed the involvement of CD133 in the architecture and dynamics of plasma membrane protrusions, such as microvilli and cilia, including the release of extracellular vesicles, as well as in various signaling pathways, which may be regulated in part by posttranslational modifications of CD133 and its interactions with a variety of proteins and lipids. Hence, CD133 appears to be a master regulator of cell signaling as its engagement in PI3K/Akt, Src-FAK, Wnt/ß-catenin, TGF-ß/Smad and MAPK/ERK pathways may explain its broad action in many cellular processes, including cell proliferation, differentiation, and migration or intercellular communication. Here, we summarize early studies on CD133, as they are essential to grasp its novel features, and describe recent evidence demonstrating that this unique molecule is involved in membrane dynamics and molecular signaling that affects various facets of tissue homeostasis and cancer development. We hope this review will provide an informative resource for future efforts to elucidate the details of CD133's molecular function in health and disease.

Mobilization of Endogenous CD34+/CD133+ Endothelial Progenitor Cells by Enhanced External Counter Pulsation for Treatment of Refractory Angina.

Adult stem cell therapy via intramyocardial injection of autologous CD34+ stem cells has been shown to improve exercise capacity and reduce angina frequency and mortality in patients with refractory angina (RA). However, the cost of such therapy is a limitation to its adoption in clinical practice. Our goal was to determine whether the less costly, less invasive, and widely accessible, FDA-approved alternative treatment for RA patients, known as enhanced external counterpulsation (EECP), mobilizes endogenous CD34+ stem cells and whether such mobilization is associated with the clinical benefits seen with intramyocardial injection. We monitored changes in circulating levels of CD34+/CD133+ and CD34+/KDR+ cells in RA patients undergoing EECP therapy and in a comparator cohort of RA patients undergoing an exercise regimen known as cardiac rehabilitation. Changes in exercise capacity in both cohorts were monitored by measuring treadmill times (TT), double product (DP) scores, and Canadian Cardiovascular Society (CCS) angina scores between pre- and post-treatment treadmill stress tests. Circulating levels of CD34+/CD133+ cells increased in patients undergoing EECP and were significant (ß = -2.38, p = 0.012) predictors of improved exercise capacity in these patients. CD34+/CD133+ cells isolated from RA patients could differentiate into endothelial cells, and their numbers increased during EECP therapy. Our results support the hypothesis that mobilized CD34+/CD133+ cells repair vascular damage and increase collateral circulation in RA patients. They further support clinical interventions that can mobilize adult CD34+ stem cells as therapy for patients with RA and other vascular diseases.

Interconnection of CD133 Stem Cell Marker with Autophagy and Apoptosis in Colorectal Cancer.

CD133 protein expression is observable in differentiated cells, stem cells, and progenitor cells within normal tissues, as well as in tumor tissues, including colorectal cancer cells. The CD133 protein is the predominant cell surface marker utilized to detect cancer cells exhibiting stem cell-like characteristics. CD133 alters common abnormal processes in colorectal cancer, such as the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/ß-catenin pathways. Autophagy is a cellular self-digestion mechanism that preserves the intracellular milieu and plays a dual regulatory role in cancer. In cancer cells, apoptosis is a critical cell death mechanism that can impede cancer progression. CD133 can modulate autophagy and apoptosis in colorectal cancer cells via several signaling pathways; hence, it is involved in the regulation of these intricate processes. This can be an explanation for why CD133 expression is associated with enhanced cellular self-renewal, migration, invasion, and survival under stress conditions in colorectal cancer. The purpose of this review article is to explain the complex relationship between the CD133 protein, apoptosis, and autophagy. We also want to highlight the possible ways that CD133-mediated autophagy may affect the apoptosis of colorectal cancer cells. Targeting the aforementioned mechanisms may have a significant therapeutic role in eliminating CD133-positive stem cell-phenotype colorectal cancer cells, which can be responsible for tumor recurrence.

CD26 Is Differentially Expressed throughout the Life Cycle of Infantile Hemangiomas and Characterizes the Proliferative Phase.

Infantile hemangiomas (IHs) are benign vascular neoplasms of childhood (prevalence 5-10%) due to the abnormal proliferation of endothelial cells. IHs are characterized by a peculiar natural life cycle enclosing three phases: proliferative (≤12 months), involuting (≥13 months), and involuted (up to 4-7 years). The mechanisms underlying this neoplastic disease still remain uncovered. Twenty-seven IH tissue specimens (15 proliferative and 12 involuting) were subjected to hematoxylin and eosin staining and a panel of diagnostic markers by immunohistochemistry. WT1, nestin, CD133, and CD26 were also analyzed. Moreover, CD31pos/CD26pos proliferative hemangioma-derived endothelial cells (Hem-ECs) were freshly isolated, exposed to vildagliptin (a DPP-IV/CD26 inhibitor), and tested for cell survival and proliferation by MTT assay, FACS analysis, and Western blot assay. All IHs displayed positive CD31, GLUT1, WT1, and nestin immunostaining but were negative for D2-40. Increased endothelial cell proliferation in IH samples was documented by ki67 labeling. All endothelia of proliferative IHs were positive for CD26 (100%), while only 10 expressed CD133 (66.6%). Surprisingly, seven involuting IH samples (58.3%) exhibited coexisting proliferative and involuting aspects in the same hemangiomatous lesion. Importantly, proliferative areas were characterized by CD26 immunolabeling, at variance from involuting sites that were always CD26 negative. Finally, in vitro DPP-IV pharmacological inhibition by vildagliptin significantly reduced Hem-ECs proliferation through the modulation of ki67 and induced cell cycle arrest associated with the upregulation of p21 protein expression. Taken together, our findings suggest that CD26 might represent a reliable biomarker to detect proliferative sites and unveil non-regressive IHs after a 12-month life cycle.

Characterization of EpCAM-Positive and EpCAM-Negative Tumor Cells in Early-Stage Breast Cancer.

Most studies on CTCs have focused on isolating cells that express EpCAM. In this study, we emphasize the presence of EpCAM-negative and EpCAMlow CTCs, in addition to EpCAMhigh CTCs, in early BC. We evaluated stem cell markers (CD44/CD24 and CD133) and EMT markers (N-cadherin) in each subpopulation. Our findings indicate that all stemness variants were present in both EpCAMhigh and EpCAM-negative CTCs, whereas only one variant of stemness (nonCD44+CD24-/CD133+) was observed among EpCAMlow CTCs. Nearly all EpCAMhigh CTCs were represented by CD133+ stem cells. Notably, the hybrid EMT phenotype was more prevalent among EpCAM-negative CTCs. scRNA-seq of isolated CTCs and primary tumor partially confirmed this pattern. Therefore, further investigation is imperative to elucidate the prognostic significance of EpCAM-negative and EpCAMlow CTCs.