Correlation of microscopic tumor extension with tumor microenvironment in esophageal cancer patients.

OBJECTIVE: In the era of image-guided adaptive radiotherapy, definition of the clinical target volume (CTV) is a challenge in various solid tumors, including esophageal cancer (EC). Many tumor microenvironmental factors, e.g., tumor cell proliferation or cancer stem cells, are hypothesized to be involved in microscopic tumor extension (MTE). Therefore, this study assessed the expression of FAK, ILK, CD44, HIF-1α, and Ki67 in EC patients after neoadjuvant radiochemotherapy followed by tumor resection (NRCHT+R) and correlated these markers with the MTE. METHODS: Formalin-fixed paraffin-embedded tumor resection specimens of ten EC patients were analyzed using multiplex immunofluorescence staining. Since gold fiducial markers had been endoscopically implanted at the proximal and distal tumor borders prior to NRCHT+R, correlation of the markers with the MTE was feasible. RESULTS: In tumor resection specimens of EC patients, the overall percentages of FAK+, CD44+, HIF-1α+, and Ki67+ cells were higher in tumor nests than in the tumor stroma, with the outcome for Ki67+ cells reaching statistical significance (p < 0.001). Conversely, expression of ILK+ cells was higher in tumor stroma, albeit not statistically significantly. In three patients, MTE beyond the fiducial markers was found, reaching up to 31 mm. CONCLUSION: Our findings indicate that the overall expression of FAK, HIF-1α, Ki67, and CD44 was higher in tumor nests, whereas that of ILK was higher in tumor stroma. Differences in the TME between patients with residual tumor cells in the original CTV compared to those without were not found. Thus, there is insufficient evidence that the TME influences the required CTV margin on an individual patient basis. TRIAL REGISTRATION NUMBER AND DATE: BO-EK-148042017 and BO-EK-177042022 on 20.06.2022, DRKS00011886, https://drks.de/search/de/trial/DRKS00011886 .

Molecular mechanisms of Codonopsis pilosula in inhibiting hepatocellular carcinoma growth and metastasis.

BACKGROUND: Liver cancer, one of the most common types of cancer worldwide, accounts for millions of cases annually. With its multi-target and wide-ranging therapeutic effects, traditional Chinese medicine has emerged as a potential approach for treating various tumors. Codonopsis pilosula, a traditional herb, is known for its anti-inflammatory and antioxidant properties. In this study, we investigated the potential molecular mechanisms of Codonopsis pilosula in regulating the inhibition of CDK1 and the modulation of PDK1/ß-catenin, which are involved in hepatocellular carcinoma growth and metastasis. STUDY DESIGN/METHODS: Firstly, we screened the active chemical constituents of Codonopsis pilosula and identified their respective target proteins using the Herb database. Then, we applied the GeneCards database and transcriptome sequencing analysis to screen for critical genes associated with the occurrence and development of liver cancer. The intersection of the target proteins and disease-related genes was used to determine the potential targets of Codonopsis pilosula in hepatocellular carcinoma. Protein-protein interaction analysis and GO/KEGG analysis were subsequently performed to uncover the pathways through which Codonopsis pilosula acts on liver cancer. The Huh-7 cell line, exhibiting the highest sensitivity to Codonopsis pilosula polysaccharide solution (CPP) intervention, was chosen for subsequent studies. Cell viability was evaluated using the CCK-8 assay, colony formation assay was conducted to determine cell proliferation capacity, flow cytometry was used to analyze cell cycle, TUNEL staining was performed to assess cell apoptosis, scratch assay was carried out to evaluate cell migration ability, the expression of EMT-related proteins was detected and analyzed, and cell sphere formation assay was conducted to investigate cell stemness. Finally, a liver cancer animal model was established, and different doses of CPP were administered via gavage the next day. The expression levels of CDK1, PDK1, and ß-catenin in mouse liver tissues were detected and analyzed, immunohistochemistry staining was performed to assess the expression of tumor cell proliferation-related proteins Ki67 and PCNA in mouse xenografts, and TUNEL staining was carried out to evaluate cell apoptosis in mouse liver tissues. After intervention with CDK1 expression, the expression levels of CDK1, PDK1, and ß-catenin proteins and mRNA in each group of cells were detected using Western blot and RT-qPCR. RESULTS: Through network pharmacology analysis, transcriptome sequencing, and bioinformatics analysis, 35 target genes through which Codonopsis pilosula acts on liver cancer were identified. Among them, CDK1, with the highest degree in the PPI network, was considered an essential target protein for Codonopsis pilosula in treating liver cancer. In vitro cell experiments revealed that CPP could inhibit the expression of CDK1/PDK1/ß-catenin signaling axis factors, suppress cell proliferation, decrease cell migration ability, influence the EMT process, and reduce cell stemness by inhibiting CDK1 and affecting the PDK1/ß-catenin signaling axis. Similarly, in vivo experiments demonstrated that CPP could regulate the CDK1/PDK1/ß-catenin signaling axis, inhibit tumor growth, and induce cell apoptosis. CONCLUSION: Codonopsis pilosula may inhibit hepatocellular carcinoma growth by suppressing CDK1 and affecting the PDK1/ß-catenin signaling axis, limiting cell EMT and reducing cell stemness. These findings provide insights into the potential therapeutic role of Codonopsis pilosula in liver cancer.

[Is it possible to detect surface antigen CD133 on patient-derived glioblastoma continuous cell cultures using fluorescent aptamers?] / Vozmozhna li detektsiya poverkhnostnogo antigena CD133 na perevivaemykh kul'turakh kletok glioblastomy patsientov s pomoshch'yu fluorestsentnykh aptamerov?

Theranostics combines diagnostics and therapeutic exposure. Regarding glioblastomas, theranostics solves the problem of detecting and destroying tumor stem cells resistant to irradiation and chemotherapy and causing tumor recurrence. Transmembrane surface antigen CD133 is considered as a potential marker of tumor stem cells. OBJECTIVE: To detect CD133 in patient-derived glioblastoma continuous cell cultures using fluorescence microscopy and modified aptamers (molecular recognition elements) anti-CD133. MATERIAL AND METHODS: To detect CD133, we used mousey fluorescence monoclonal antibodies anti-CD133 MA1-219, FAM-modified DNA aptamers anti-CD133 AP-1-M and Cs5. Non-aptamer DNA oligonucleotide NADO was used as a negative control. Detection was performed for three samples of patient-derived glioblastoma continuous cell cultures coded as 1548, 1721 and 1793. RESULTS: MA1-219 antibodies brightly stained cell culture 1548, to a lesser extent - 1721. There was diffuse staining of cell culture 1793. Cs5-FAM aptamer stained cells in a similar way, but much weaker. AP-1-M-FAM aptamer interacted with cells even weaker and diffusely stained only cell culture 1793. Non-aptamer NADO did not stain cell culture 1548 and very weakly diffusely stained cell culture 1793. CONCLUSION: For both molecular recognition elements (MA1-219 antibody and Cs5 aptamer), 3 cell culture samples can be arranged in the following order possibly reflecting CD133 status decrease: strong signal for cell culture 1548, much weaker for 1721, even weaker for 1793. Only cell culture 1548 can be considered CD133 positive with combination of Cs5+ and NADO signals. Cell culture 1793 is CD133 false positive with combination of Cs5+ and NADO+ signals.

[Advances in the biological characteristics and stem maintenance mechanisms of tumor stem cells].

Tumor is one of the most serious diseases that threaten human health and social development, and it is the second most common cause of death worldwide. The latest statistics show that malignant tumors have surpassed cardiovascular disease as the leading cause of death in developed countries. Drug resistance, metastasis, and recurrence of tumors continue to present urgent challenges in clinical treatment. Tumor stem cells (TSCs) are a specific subset of cells that possess high capabilities of self-renewal, differentiation potential, tumorigenicity and drug resistance. They are resistant to non-specific treatment methods such as chemotherapy and radiotherapy, and play a crucial role in tumor initiation, metastasis, drug resistance, and recurrence. The surface markers, stemness maintenance mechanisms, microenvironment, and metabolic reprogramming of TSCs have become areas of intense research focus. The latest research results provide novel targets and strategies for the identification of TSCs and targeted therapy. This paper reviews the surface markers (CD133, CD44, etc.), self-renewal and epithelial mesenchymal transition (EMT) signaling pathways (Wnt/ß-catenin, Hedgehog, etc.), microenvironment characteristics, metabolic reprogramming (glycolysis, oxidative phosphorylation, etc.) and their roles in the initiation, development, metastasis and drug resistance of TSCs.

A novel approach for transforming breast cancer stem cells into endothelial cells.

Tumor vascular endothelial cells play a pivotal in the tumor microenvironment, influencing the proliferation, invasion, and metastasis of tumor progression. The present study investigated a novel method for inducing the transformation of breast cancer stem cells into endothelial cells, providing a cellular model investigating anti-angiogenic mechanisms in vitro. The breast cancer cell line MCF-7 was used, and the expression of CD133 was initially detected using flow cytometry. CD133+ breast cancer cells were purified using immunomagnetic bead sorting technology, yielding an MCF-7CD133+ subpopulation. The proliferation ability of these cells was assessed using an MTT assay, while their microsphere formation ability was evaluated using a microsphere formation assay. Post-transformation in an optimized endothelial cell culture medium, expression of endothelial cell markers CD31 and CD105 were detected using flow cytometry. Endothelial cell tube formation assays and DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) assays were employed to analyze the endothelial cell function of the MCF-7CD133+ cells. MDM2/CEN12 gene amplification was detected through fluorescence in situ hybridization (FISH). The MCF-7 breast cancer cell line exhibited 1.7±0.3% trace cells expressing the stem cell surface marker CD133. After anti-CD133 immunomagnetic bead sorting, MCF-7CD133+ and MCF-7CD133- subpopulation cells were obtained, with CD133 expression rates of 85.6±2.8 and 0.18±0.08%, respectively. MTT assay results demonstrated that, after 7 days, the proliferation rate of MCF-7CD133+ cells was significantly higher compared with MCF-7CD133- cells. MCF-7CD133+ subpopulation cells displayed strong stem cell characteristics, growing in suspension in serum-free media and forming tumor cell spheres. In contrast, MCF-7CD133- cells failed to form microspheres. After culturing cells in endothelial cell differentiation and maintenance media, the percentage of MCF-7CD133+ cells before and after endothelial cell culture was 0.3±0.16 and 81.4±8.37% for CD31+ cells and 0.2±0.08 and 83.8±7.24% for CD105+ cells, respectively. Vascular-like structure formation and Ac-LDL phagocytosis with red fluorescence in the tube formation assays confirmed endothelial cell function in the MCF-7CD133+ cells. FISH was used to verify MDM2/CEN12 gene amplification in the induced MCF-7CD133+ cells, indicating tumor cell characteristics. The modified endothelial cell transformation medium effectively induced differentiated tumor stem cells to express vascular endothelial cell markers and exhibit endothelial functions, ideal for in vitro anti-angiogenesis research.

Distinctive mesenchymal-like neurofibroma stem cells shape NF1 clinical phenotypes controlled by BDNF microenvironment.

BACKGROUND: Neurofibroma type I (NF1) often presents with multiple clinical phenotypes due to mutations of NF1 gene. The aim of this study was to determine the phenotypic and therapeutic relevance of tumor microenvironment in NF1 patients. METHODS: Tumor stem cells (TSCs) from NF1 were isolated and cultured using fluorescence activated cell sorting (FACS) and colony formation experiments. Then, flow cytometry was used to detect the surface markers, osteogenic and adipogenic differentiation were performed as well. Its tumorigenesis ability was confirmed by subcutaneous tumorigenesis in nude mice. Immunohistochemical staining was performed on neurofibroma tissues from the head and trunk with different phenotypes. The expression of BDNF in neurofibroma tissues was detected by Elisa and immunohistochemical staining. Western Blotting was used to detect the expression of p38 MAPK pathway in TSCs. The effect of BDNF neutralizing antibody on the tumorigenesis of TSCs was observed. RESULTS: Herein, we advocate that NF1 contain a new subgroup of mesenchymal-like neurofibroma stem cells (MNSCs). Such colony-forming MNSCs preserved self-renewal, multiple differentiation and tumorigenic capabilities. More interestingly, the MNSCs isolated from neurofibroma tissues of the same patient with different phenotypes presented site-specific capabilities. Moreover, different levels of brain-derived neurotrophic factor (BDNF) in neurofibroma tissues can impact the MNSCs by activating the TrkB/p38 MAPK pathway. Systemic administration of BDNF neutralizing antibodies inhibited MNSCs' characteristics. CONCLUSIONS: We demonstrated that BDNF can modulate MNSCs and thereby controlling different tumor phenotypes between the head and trunk regions. Application of BDNF neutralizing antibodies may inhibit p38 MAPK pathway, therefore providing a promising strategy for managing NF1.

Identifying a Novel Eight-NK Cell-related Gene Signature for Ovarian Cancer Prognosis Prediction.

BACKGROUND: Ovarian cancer (OVC) is the most common and costly tumor in the world with unfavorable overall survival and prognosis. This study is aimed to explore the prognostic value of natural killer cells related genes for OVC treatment. METHODS: RNA-seq and clinical information were acquired from the TCGA-OVC dataset (training dataset) and the GSE51800 dataset (validation dataset). Genes linked to NK cells were obtained from the immPort dataset. Moreover, ConsensusClusterPlus facilitated the screening of molecular subtypes. Following this, the risk model was established by LASSO analysis, and immune infiltration and immunotherapy were then detected by CIBERSORT, ssGSEA, ESTIMATE, and TIDE algorithms. RESULTS: Based on 23 NK cell-related genes with prognosis, TCGA-OVC samples were classified into two clusters, namely C1 and C2. Of these, C1 had better survival outcomes as well as enhanced immune infiltration and tumor stem cells. Additionally, it was more suitable for immunotherapy and was also sensitive to traditional chemotherapy drugs. The eight-gene prognosis model was constructed and verified via the GSE51800 dataset. Additionally, a high infiltration level of immune cells was observed in low-risk patients. Low-risk samples also benefited from immunotherapy and chemotherapy drugs. Finally, a nomogram and ROC curves were applied to validate model accuracy. CONCLUSION: The present study identified a RiskScore signature, which could stratify patients with different infiltration levels, immunotherapy, and chemotherapy drugs. Our study provided a basis for precisely evaluating OVC therapy and prognosis.

The Nexus of Inflammation-Induced Epithelial-Mesenchymal Transition and Lung Cancer Progression: A Roadmap to Pentacyclic Triterpenoid-Based Therapies.

Lung cancer is the leading cause of cancer-related death worldwide. Its high mortality is partly due to chronic inflammation that accompanies the disease and stimulates cancer progression. In this review, we analyzed recent studies and highlighted the role of the epithelial-mesenchymal transition (EMT) as a link between inflammation and lung cancer. In the inflammatory tumor microenvironment (iTME), fibroblasts, macrophages, granulocytes, and lymphocytes produce inflammatory mediators, some of which can induce EMT. This leads to increased invasiveness of tumor cells and self-renewal of cancer stem cells (CSCs), which are associated with metastasis and tumor recurrence, respectively. Based on published data, we propose that inflammation-induced EMT may be a potential therapeutic target for the treatment of lung cancer. This prospect is partially realized in the development of EMT inhibitors based on pentacyclic triterpenoids (PTs), described in the second part of our study. PTs reduce the metastatic potential and stemness of tumor cells, making PTs promising candidates for lung cancer therapy. We emphasize that the high diversity of molecular mechanisms underlying inflammation-induced EMT far exceeds those that have been implicated in drug development. Therefore, analysis of information on the relationship between the iTME and EMT is of great interest and may provide ideas for novel treatment approaches for lung cancer.

Esophageal cancer stem cells reduce hypoxia-induced apoptosis by inhibiting the GRP78-perk-eIF2α-ATF4-CHOP pathway in vitro.

Background: Due to the abnormal angiogenesis, cancer stem cells (CSCs) in esophageal cancer (EC) have the characteristics of a hypoxic microenvironment. However, they can resist hypoxia-induced apoptosis. the molecular mechanism underlying the resistance of esophageal CSCs to hypoxia-induced apoptosis is currently unclear. Therefore, this study will investigate the molecular mechanism based on CHOP-mediated endoplasmic reticulum stress. Methods: CD44+CD24- cells in EC9706 cells were screened by fluorescence-activated cell sorting (FACS). To clarify which apoptosis pathway esophageal CSCs resist hypoxia-induced cell apoptosis through, the effects of hypoxia on apoptosis were detected by nuclear staining, flow cytometry, and JC-1 reagent, the effects of hypoxia on the expression of apoptosis-related proteins were detected by western blotting (WB) assay and quantitative polymerase chain reaction (qPCR) assay. To clarify the mechanisms of CD44+CD24- cells resistance to hypoxia-induced apoptosis is achieved by inhibiting the activation of endoplasmic reticulum stress (ERS) pathway, silenced CHOP and PERK cell lines of EC9706 cells and overexpressed CHOP and PERK cell lines of CD44+CD24- cells were constructed, the effects of hypoxia on apoptosis, cell cycle, and mitochondrial membrane potential were detected by flow cytometry and JC-1 reagent. WB assay and qPCR assay were used to detect the expressions of apoptosis-related proteins and ERS-related proteins. Results: Hypoxia significantly induce apoptosis and cycle arrest of EC9706 cells (P<0.05), but did not affect apoptosis and cycle of CD44+CD24- cells (P>0.05). Hypoxia considerably induced the activation of mitochondrial and ERS apoptosis pathways in EC9706 cells (P<0.05), but did not affect Fas receptor apoptosis pathways (P>0.05). The three apoptosis pathways were not affected by hypoxia in CD44+CD24- cells (P>0.05). Silencing the CHOP and PERK gene inhibited hypoxia-induced apoptosis of EC9706 cells (P<0.05). CHOP and PERK overexpression promoted hypoxia-induced apoptosis of CD44+CD24- cells (P<0.05), whereas mitochondrial membrane permeability inhibitors inhibited hypoxia-induced apoptosis of CD44+CD24- cells overexpressed CHOP gene. Conclusions: CD44+CD24- tumor stem cells in EC resist to hypoxia-induced apoptosis by the inhibition of ERS-mediated mitochondrial apoptosis pathway, which suggested that ERS pathway can serve as a potential target for reducing EC treatment resistance in clinical treatment.

Response to primary chemoradiotherapy of locally advanced oropharyngeal carcinoma is determined by the degree of cytotoxic T cell infiltration within tumor cell aggregates.

Background: Effective anti-tumor immune responses are mediated by T cells and require organized, spatially coordinated interactions within the tumor microenvironment (TME). Understanding coordinated T-cell-behavior and deciphering mechanisms of radiotherapy resistance mediated by tumor stem cells will advance risk stratification of oropharyngeal cancer (OPSCC) patients treated with primary chemoradiotherapy (RCTx). Methods: To determine the role of CD8 T cells (CTL) and tumor stem cells for response to RCTx, we employed multiplex immunofluorescence stains on pre-treatment biopsy specimens from 86 advanced OPSCC patients and correlated these quantitative data with clinical parameters. Multiplex stains were analyzed at the single-cell level using QuPath and spatial coordination of immune cells within the TME was explored using the R-package Spatstat. Results: Our observations demonstrate that a strong CTL-infiltration into the epithelial tumor compartment (HR for overall survival, OS: 0.35; p<0.001) and the expression of PD-L1 on CTL (HR: 0.36; p<0.001) were both associated with a significantly better response and survival upon RCTx. As expected, p16 expression was a strong predictor of improved OS (HR: 0.38; p=0.002) and correlated with overall CTL infiltration (r: 0.358, p<0.001). By contrast, tumor cell proliferative activity, expression of the tumor stem cell marker CD271 and overall CTL infiltration, regardless of the affected compartment, were not associated with response or survival. Conclusion: In this study, we could demonstrate the clinical relevance of the spatial organization and the phenotype of CD8 T cells within the TME. In particular, we found that the infiltration of CD8 T cells specifically into the tumor cell compartment was an independent predictive marker for response to chemoradiotherapy, which was strongly associated with p16 expression. Meanwhile, tumor cell proliferation and the expression of stem cell markers showed no independent prognostic effect for patients with primary RCTx and thus requires further study.

Electromagnetic fields regulate calcium-mediated cell fate of stem cells: osteogenesis, chondrogenesis and apoptosis.

Electromagnetic fields (EMF) are increasing in popularity as a safe and non-invasive therapy. On the one hand, it is widely acknowledged that EMF can regulate the proliferation and differentiation of stem cells, promoting the undifferentiated cells capable of osteogenesis, angiogenesis, and chondroblast differentiation to achieve bone repair purpose. On the other hand, EMF can inhibit tumor stem cells proliferation and promote apoptosis to suppress tumor growth. As an essential second messenger, intracellular calcium plays a role in regulating cell cycle, such as proliferation, differentiation and apoptosis. There is increasing evidence that the modulation of intracellular calcium ion by EMF leads to differential outcomes in different stem cells. This review summarizes the regulation of channels, transporters, and ion pumps by EMF-induced calcium oscillations. It furtherly discusses the role of molecules and pathways activated by EMF-dependent calcium oscillations in promoting bone and cartilage repair and inhibiting tumor stem cells growth.

Effects of Dendrimer-microRNA Nanoformulations against Glioblastoma Stem Cells.

Glioblastoma is a rapidly progressing tumor quite resistant to conventional treatment. These features are currently assigned to a self-sustaining population of glioblastoma stem cells. Anti-tumor stem cell therapy calls for a new means of treatment. In particular, microRNA-based treatment is a solution, which in turn requires specific carriers for intracellular delivery of functional oligonucleotides. Herein, we report a preclinical in vitro validation of antitumor activity of nanoformulations containing antitumor microRNA miR-34a and microRNA-21 synthetic inhibitor and polycationic phosphorus and carbosilane dendrimers. The testing was carried out in a panel of glioblastoma and glioma cell lines, glioblastoma stem-like cells and induced pluripotent stem cells. We have shown dendrimer-microRNA nanoformulations to induce cell death in a controllable manner, with cytotoxic effects being more pronounced in tumor cells than in non-tumor stem cells. Furthermore, nanoformulations affected the expression of proteins responsible for interactions between the tumor and its immune microenvironment: surface markers (PD-L1, TIM3, CD47) and IL-10. Our findings evidence the potential of dendrimer-based therapeutic constructions for the anti-tumor stem cell therapy worth further investigation.

Identification and gene expression profiling of human gonadotrophic pituitary adenoma stem cells.

BACKGROUND: Gonadotrophic pituitary adenoma is a major subtype of pituitary adenoma in the sellar region, but it is rarely involved in the hypersecretion of hormones into blood; thus, it is commonly regarded as "non-functioning." Its tumorigenic mechanisms remain unknown. The aim of this study was to identify human gonadotrophic pituitary adenoma stem cells (hPASCs) and explore the underlying gene expression profiles. In addition, the potential candidate genes involved in the invasive properties of pituitary adenoma were examined. METHODS: The hPASCs from 14 human gonadotrophic pituitary adenoma clinical samples were cultured and verified via immunohistochemistry. Genetic profiling of hPASCs and the matched tumor cells was performed through RNA-sequencing and subjected to enrichment analysis. By aligning the results with public databases, the candidate genes were screened and examined in invasive and non-invasive gonadotrophic pituitary adenomas using Real-time polymerase chain reaction. RESULTS: The hPASCs were successfully isolated and cultured from gonadotrophic pituitary adenoma in vitro, which were identified as positive for generic stem cell markers (Sox2, Oct4, Nestin and CD133) via immunohistochemical staining. The hPASCs could differentiate into the tumor cells expressing follicle-stimulating hormone in the presence of fetal bovine serum in the culture medium. Through RNA-sequencing, 1352 differentially expressed genes were screened and identified significantly enriched in various gene ontologies and important pathways. The expression levels of ANXA2, PMAIP1, SPRY2, C2CD4A, APOD, FGF14 and FKBP10 were significantly upregulated while FNDC5 and MAP3K4 were downregulated in the invasive gonadotrophic pituitary adenomas compared to the non-invasive ones. CONCLUSION: Genetic profiling of hPASCs may explain the tumorigenesis and invasiveness of gonadotrophic pituitary adenoma. ANXA2 may serve as a potential therapeutic target for the treatment of gonadotrophic pituitary adenoma.

Oct-4 induces cisplatin resistance and tumor stem cell-like properties in endometrial carcinoma cells.

OBJECTIVE: Research has suggested that tumor-initiating tumor stem cells are derived from normal stem cells and that tumor cells undergo progressive de-differentiation to achieve a stem cell-like state. Tumor stem cells are characterized by high proliferation ability, high plasticity, expression of multi-drug resistance proteins, and the ability to seed new tumors. Octamer-binding transcription factor 4 (Oct-4) and its activation targets are overexpressed in the tumor stem cells of various types of tumors, and this expression is associated with the pathogenesis, development, and poor prognosis of tumors. The primary objective of this study was to test if a stably transfected with Oct-4 gene cell line, RL95-2/Oct-4, has the characteristics of tumor stem cells. MATERIALS AND METHODS: Human endometrial carcinoma cells (RL95-2) were transfected with a plasmid carrying genes for Oct-4 and green fluorescent protein (GFP). The stably transfected cells, RL95-2/Oct-4, were selected using G418 and observed to express the GFP reporter gene under the control of the Oct-4 promoter. GFP expression levels of RL95-2/Oct-4 cells were measured using flow cytometry. The proliferation potential of cells was determined according to cumulative population doubling and colony-formation efficiency. Gene expression was analyzed using reverse transcription-polymerase chain reaction. RESULTS: RL95-2/Oct-4 cells not only exhibited increased expression of the three most important stem cell genes, Oct-4, Nanog, and Sox2, but also had increased expression of the endometrial tumor stem cell genes CD133 and ALDH1. Furthermore, enhanced expression of these genes in the RL95-2/Oct-4 cells was associated with higher colony-forming ability and growth rate than in parental RL95-2 cells. We also observed that cisplatin induced less cell death in RL95-2/Oct-4 cells than in RL95-2 cells, indicating that RL95-2/Oct-4 cells were more resistant to chemotherapeutic agents. CONCLUSION: The study findings contribute to investigate the effects of Oct-4 on tumor stem cell origins.

BI-907828, a novel potent MDM2 inhibitor, inhibits glioblastoma brain tumor stem cells in vitro and prolongs survival in orthotopic xenograft mouse models.

BACKGROUND: The tumor suppressor TP53 (p53) is frequently mutated, and its downstream effectors inactivated in many cancers, including glioblastoma (GBM). In tumors with wild-type status, p53 function is frequently attenuated by alternate mechanisms including amplification and overexpression of its key negative regulator, MDM2. We investigated the efficacy of the MDM2 inhibitor, BI-907828, in GBM patient-derived brain tumor stem cells (BTSCs) with different amplification statuses of MDM2, in vitro and in orthotopic xenograft models. METHODS: In vitro growth inhibition and on-target efficacy of BI-907828 were assessed by cell viability, co-immunoprecipitation assays, and western blotting. In vivo efficacy of BI-907828 treatments was assessed with qPCR, immunohistochemistry, and in intracranial xenograft models. RESULTS: BI-907828 decreases viability and induces cell death at picomolar concentrations in both MDM2 amplified and normal copy number TP53 wild-type BTSC lines. Restoration of p53 activity, including robust p21 expression and apoptosis induction, was observed in TP53 wild-type but not in TP53 mutant BTSCs. shRNA-mediated knock-down of TP53 in wild-type BTSCs abrogated the effect of BI-907828, confirming the specificity of the inhibitor. Pharmacokinetic-pharmacodynamic studies in orthotopic tumor-bearing severe combined immunodeficiency (SCID) mice demonstrated that a single 50 mg/kg p.o. dose of BI-907828 resulted in strong activation of p53 target genes p21 and MIC1. Long-term weekly or bi-weekly treatment with BI-907828 in orthotopic BTSC xenograft models was well-tolerated and improved survival both as a single-agent and in combination with temozolomide, with dose-dependent efficacy observed in the MDM2 amplified model. CONCLUSIONS: BI-907828 provides a promising new therapeutic option for patients with TP53 wild-type primary brain tumors.

Effects of co-culture of dendritic cells loaded with MAGE-A3 antigen and cytokine-induced killer cells on tumor stem cells and malignant progression of endometrial cancer / 中国临床药理学与治疗学

AIM: To explore the effects of dendritic cells (DC) and cytokine-induced killer cells (CIK) carrying melanoma-associated antigen gene A3 (MAGE-A3) on endometrial cancer tumor stem cells and malignant progression. METHODS: Human peripheral blood was collected to separate mono-nuclear cells, and DC and CIK cells were induced by cytokines, respectively. DCs were incubated with MAGE-A3 and then co-cultured with CIK, and the phenotypes of DC-CIK and MAGE-A3-DC-CIK were detected by flow cytometry; The CD133

[Molecular markers of neuro-oncogenesis in patients with glioblastoma]. / Molekulyarnye markery neiroonkogeneza pri glioblastome golovnogo mozga.

The problem of current treatment approaches to brain gliomas is short-term life expectancy in these patients. Apparently, it is required to change treatment approach via analysis of glioma stem cells rather cells with overexpression of marker genes. This review is devoted to similarities and differences between neurogenesis and neuro-oncogenesis characterized with molecular markers (CD133 as an example). The role of tumor stem cells and their relationship with neural stem cells are considered regarding development of glioma. The authors analyzed CD133 as a marker of glioma stem cells. In the future, stem cells will be important target for eradication during target therapy. A single molecular marker cannot characterize tumor stem cells as supported by CD133 studies. A set of molecular markers specific for certain cell type is required, and their combination will provide more accurate establishment of tumor stem cells.

[Protein CD133 as a tumor stem cell marker]. / Belok CD133 kak marker opukholevykh stvolovykh kletok.

The CD133 protein is a large transmembrane glycoprotein. Despite multiple studies, the role of CD133 protein in cells is still poorly understood. Nevertheless, there is an association of CD133 protein with neoplastic transformation. This review summarizes data on CD133 protein, its structure, regulation of expression, molecular interactions and representation in cells that have undergone malignant transformation. Available data suggest that CD133 may have a great potential for predicting survival in various solid tumors. This protein can also be a marker of glioma.

The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.