Levels Of Cancer Stem Cells Research Articles

Human-derived Tumor-On-Chip model to study the heterogeneity of breast cancer tissue

One of the leading causes that complicate the treatment of some malignancies, including breast cancer, is tumor heterogeneity. In addition to inter-heterogeneity and intra-heterogeneity of tumors that reflect the differences between cancer cell characteristics, heterogeneity in the tumor microenvironment plays a critical role in tumor progression and could be considered an overlooked and a proper target for the effective selection of therapeutic approaches. Due to the difficulty of completely capturing tumor heterogeneity in conventional detection methods, Tumor-on-Chip (TOC) devices with culturing patient-derived spheroids could be an appropriate alternative. In this research, human-derived spheroids from breast cancer individuals were cultured for 6 days in microfluidic devices. To compare TOC data with conventional detection methods, immunohistochemistry (IHC) and ITRAQ data were employed, and various protein expressions were validated using the transcriptomic databases. The behavior of the spheroids in the collagen matrix and the cell viability were monitored over 6 days of culture. IHC and immunocytochemistry (ICC) results revealed that inter and intra-heterogeneity of tumor spheroids are associated with HER2/ER expression. HER2 expression levels revealed a more important biomarker associated with invasion in the 3D culturing of spheroids. The expression levels of CD163 (as a marker for Ma2 macrophages) and CD44 (a marker for cancer stem cells (CSCs)) were also evaluated. Interestingly, the levels of M2a macrophages and CSCs were higher in triple-negative specimens and samples that showed higher migration and invasion. Cell density and extracellular matrix (ECM) stiffness were also important factors affecting the migration and invasion of the spheroids through the matrix. Among these, rigid ECM revealed a more crucial role than cell density. To sum up, these research findings demonstrated that human-derived spheroids from breast cancer specimens in microfluidic devices provide a dynamic condition for predicting tumor heterogeneity in patients, which can help move the field forward for better and more accurate therapeutic strategies.

Abstract 5120: Direct targeting of the COMPASS complex inhibits neuroblastoma growth by inhibiting cancer stem cells

Abstract Epigenetic regulator COMPASS Complex, a histone H3K4 methyltransferase has recently been shown to be involved in multiple cancers and attaining focus as a novel therapeutic target. High-risk neuroblastoma (NB) is a heterogeneous pediatric cancer with high mortality and relapse rates. The relapse of refractory cancer is shown to be driven by cancer stem cells (CSCs), which are maintained by epigenetic malfunctions. In the present study, we determined the effects of inhibiting the COMPASS complex on NB CSCs and overall NB growth. A high-throughput shRNA screening of methyltransferases and demethylases reveals the role of COMPASS complex partners MLL1 and Menin in NB proliferation. We found that both MLL1 and Menin are overexpressed in our previously identified CD114+ NB CSCs. Further, MEN1 (gene coding Menin) expression showed an inverse correlation with overall NB patient survival and cancer progression in a comprehensive analysis of 1235 primary NB patient data. Our ChIP results show that the CSF3R gene which codes for CD114 and maintains CD114+ NB CSCs, is regulated by the presence of high H3K4me3 activating marks. Further, we used MI-503 to inhibit MLL1-Menin interaction and also developed MEN1 stable knockdown and found that both strategies significantly inhibit H3K4me3 activity in NB, particularly at the CSF3R gene promoter. This led to decreased CSF3R expression and NB CSC levels. MI-503 significantly inhibits proliferation in different NB cell lines and primary patient-derived xenograft (PDX) lines, with minimal effect on non-cancerous fibroblasts. MI-503 significantly inhibits NB cell cycle progression at the S phase, induces apoptosis, and inhibits NB 3D spheroid growth and development. Interestingly, we overserved differential selectivity of MI-503 in inducing apoptosis and in blocking the cell cycle in NB CSCs in contrast to non-CSCs. MEN1 knockdown also inhibits NB proliferation and 3D spheroid growth. Furthermore, MI-503 in a dose-dependent manner synergistically sensitizes NB to chemotherapy doxorubicin and significantly inhibits NB proliferation and spheroid growth compared to either agent alone. Further, by using the NB xenograft in vivo tumor model, we established that MI-503 significantly inhibits NB tumor growth and tumor metastasis by directly inhibiting tumor NB CSCs, without any observed toxicities. Similar results of significantly reduced tumor growth, metastasis, and NB CSCs were shown by the MEN1 knockdown in vivo. Overall, our data show that the MLL1-Menin of the COMPASS complex directly activates the CSF3R gene to maintain NB CSCs and drive NB pathogenesis. Our data show that MI-503 or MEN1 knockdown leads to significant inhibition of NB growth by directly inhibiting NB CSCs in both in vitro and in vivo tumor models. Further developing these epigenetic-based therapeutic strategies and combining them with current therapies will pave the way for effectively managing NB. Citation Format: Rameswari Chilamakuri, Saurabh Agarwal. Direct targeting of the COMPASS complex inhibits neuroblastoma growth by inhibiting cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5120.

Identification of novel potential homologous repair deficiency-associated genes in pancreatic adenocarcinoma via WGCNA coexpression network analysis and machine learning

ABSTRACT Homologous repair deficiency (HRD) impedes double-strand break repair, which is a common driver of carcinogenesis. Positive HRD status can be used as theranostic markers of response to platinum- and PARP inhibitor-based chemotherapies. Here, we aimed to fully investigate the therapeutic and prognostic potential of HRD in pancreatic adenocarcinoma (PAAD) and identify effective biomarkers related to HRD using comprehensive bioinformatics analysis. The HRD score was defined as the unweighted sum of the LOH, TAI, and LST scores, and it was obtained based on the previous literature. To characterize PAAD immune infiltration subtypes, the “ConsensusClusterPlus” package in R was used to conduct unsupervised clustering. A WGCNA was conducted to elucidate the gene coexpression modules and hub genes in the HRD-related gene module of PAAD. The functional enrichment study was performed using Metascape. LASSO analysis was performed using the “glmnet” package in R, while the random forest algorithm was realized using the “randomForest” package in R. The prognostic variables were evaluated using univariate Cox analysis. The prognostic risk model was built using the LASSO approach. ROC curve and KM survival analyses were performed to assess the prognostic potential of the risk model. The half-maximal inhibitory concentration (IC50) of the PARP inhibitors was estimated using the “pRRophetic” package in R and the Genomics of Drug Sensitivity in Cancer database. The “rms” package in R was used to create the nomogram. A high HRD score indicated a poor prognosis and an advanced clinical process in PAAD patients. PAAD tumors with high HRD levels revealed significant T helper lymphocyte depletion, upregulated levels of cancer stem cells, and increased sensitivity to rucaparib, Olaparib, and veliparib. Using WGCNA, 11 coexpression modules were obtained. The red module and 122 hub genes were identified as the most correlated with HRD in PAAD. Functional enrichment analysis revealed that the 122 hub genes were mainly concentrated in cell cycle pathways. One novel HRD-related gene signature consisting of CKS1B, HJURP, and TPX2 were screened via LASSO analysis and a random forest algorithm, and they were validated using independent validation sets. No direct association between HRD and CKS1B, HJURP, or TPX2 has not been reported in the literature so far. Thus, these findings indicated that CKS1B, HJURP, and TPX2 have potential as diagnostic and prognostic biomarkers for PAAD. We constructed a novel HRD-related prognostic model that provides new insights into PAAD prognosis and immunotherapy. Based on bioinformatics analysis, we comprehensively explored the therapeutic and prognostic potential of HRD in PAAD. One novel HRD-related gene signature consisting of CKS1B, HJURP, and TPX2 were identified through the combination of WGCNA, LASSO analysis and a random forest algorithm. A novel HRD-related risk model that can predict clinical prognosis and immunotherapeutic response in PAAD patients was constructed.

The Combination of PD-L1 and CTLA-4 Suppression Significantly Decreased the Expression Levels of Cancer Stem Cell Factors in the Pancreatic Cancer Cell Line

Background: It is thought that a limited number of aberrant cancer stem cells (CSCs), which encourage carcinogenesis, tumor metastasis, and treatment resistance, are the cause of pancreatic cancer, a disease with a high mortality rate. Increasing evidence shows that CSCs use immunosuppressive properties to avoid immune system identification. In Pancreatic cancer, the therapeutic consequences of the relationship between the expression of immune checkpoints such as programmed death receptor ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the presence of CSCs are poorly known. As a result, in the present investigation, we examined how the expression levels of CSCs were affected by PD-L1 and CTLA-4 knockdown using specific siRNA. Methods: Independently and together, PD-L1 and CTLA-4 siRNA were transfected into MIA PaCa-2 cells. RNA extraction and cDNA synthesis were then performed. Finally, using qRT-PCR, the gene expression levels of CSCs markers, including Nanog, CD133, CD44, and Oct-4, in transfected groups were measured. Results: In the MIA PaCa-2 cell line, siRNA-mediated inhibition of PD-L1 and CTLA-4 decreased the expression of CSC factors. Moreover, combined suppression of these two stemness-related immune checkpoints significantly decreased Nanog, CD133, CD44, and Oct-4 expression compared to inhibition of PD-L1 and CTLA-4 separately. Conclusion: Considering that the combination of PD-L1 and CTLA-4 suppression using siRNA significantly decreased the expression levels of CSCs factors, this approach may be regarded as an effective therapy for this cancer.

Clinicopathological significance of cancer stem cell marker CD44/SOX2 in esophageal squamous cell carcinoma (ESCC) patients and construction of a nomogram to predict overall survival.

Esophageal squamous cell carcinoma (ESCC), a prevalent malignancy within the upper gastrointestinal system, is characterized by its unfavorable prognosis and the absence of specific indicators for outcome prediction and high-risk case identification. In our research, we examined the expression levels of cancer stem cells (CSCs), markers CD44/SOX2 in ESCC, scrutinized their association with clinicopathological parameters, and developed a predictive nomogram model. This model, which incorporates CD44/SOX2, aims to forecast the overall survival (OS) of patients afflicted with ESCC. Immunohistochemistry was utilized to detect the expression levels of CD44 and SOX2 in both cancerous and paracancerous tissues of 68 patients with ESCC. The correlation between CD44/SOX2 expression and clinicopathological parameters was subsequently analyzed. Factors impacting the prognosis of ESCC patients were assessed through univariate and multivariate Cox regression analyses. Leveraging the results of these multivariate regression analyses, a nomogram prognostic model was established to provide individualized predictions of ESCC patient survival outcomes. The predictive accuracy of the nomogram prognostic model was evaluated using the consistency index (C-index) and calibration curves. The expression levels of CD44 were markedly elevated in the tumor tissues of ESCC patients. Similarly, SOX2 was significantly overexpressed in the tumor tissues of ESCC patients. The positive expression of SOX2 in ESCC demonstrated a strong correlation with both the pathological T-stage and the presence of carcinoembryonic antigen. CD44 and SOX2 co-positive expression was significantly associated with the pathological T-stage and tumor node metastasis (TNM) stage. Furthermore, ESCC patients exhibiting CD44-positive expression in their tumor tissue generally had a more adverse prognosis. The co-expression of CD44 and SOX2 resulted in a grimmer prognosis compared to patients with other combinations. Multivariate Cox regression analysis identified the co-expression of CD44 and SOX2, the pathological T-stage, and lymph node metastasis as independent prognostic indicators for ESCC patients. The three identified variables were subsequently incorporated into a nomogram for predicting OS. The C-index of the measurement model and the area under the curve of the subjects' work characteristics showed good individual prediction. This prognostic model stratified patients into low- and high-risk categories. Analysis revealed that the 5-year OS rate was significantly higher in the low-risk group compared to the high-risk group. Elevated CD44 levels, indicative of CSC presence, are intimately linked with the oncogenesis of ESCC and are strongly predictive of unfavorable patient outcomes. Concurrently, the SOX2 gene exhibits a heightened expression in ESCC, markedly accelerating tumor progression and fostering more extensive disease infiltration. The co-expression of CD44 and SOX2 correlates significantly with ESCC patient prognosis, serving as a reliable, independent prognostic marker. Our constructed nomogram, incorporating CD44/SOX2 expression, enhances the prediction of OS and facilitates risk stratification in ESCC patients.

Overall survival in colon cancer patients with different levels of circulating tumor cells and feasibility of enhancing its prognostic value

Development of personalized approaches to diagnosis, treatment and prognosis of colon cancer (CC) still remains challenging. Levels of circulating tumor (CTC) and cancer stem cells (CSC) are promising non-invasive prognostic factors. Our aim was to assess the overall survival (OS) of patients with stage II–IV CC with different levels of CTCs as well as to enhance their prognostic value by additionally determining the level of CD44+ CSCs. Material and methods. The study included 299 patients with stage II–IV CC. All patients underwent surgery followed by adjuvant chemotherapy (FOLFOX). patients with stage IV CC with resectable liver metastases underwent simultaneous resection of the primary tumor and liver metastases, followed by FOLFOX chemotherapy. the proportion of CTCs was evaluated before surgery using Veridex CellSearch™, and the level of CD44+ CSCs was determined in the tissue of the removed tumor by the IHC method. OS was studied in patients with different CTC levels, cumulative OS was calculated by Kaplan–Meier`s method. prognostic algorithm was designed by logistic regression analysis and cox proportional hazards model. Results. OS was found to be lower in patients with higher CTC levels divided into ranges: 0, 1–3, 4–9, ≥10 (χ2=11.59, p=0.009); thus enabling us to use it for prognosis. its prognostic value is enhanced by estimation of CD44+ CSC in tumor. Statistically significant conjugation between CTC and CD44+ ranges was found. an increase in CTC level by 1 range resulted in the increase in the risk of fatal outcome by 1.58 times (р=0.002); the additive increase in CD44+ expression ≥10 % resulted in the increase in the risk of fatal outcome by 7.2 times (p<0.001). For individual risk assessment, a model for calculating the prognostic coefficient K with high diagnostic sensitivity and specificity was developed, and its mathematical expression was proposed. the value of K≥0.411 indicates a high risk of adverse outcome. Conclusion. The prognostic algorithm for the risk of unfavorable outcome of patients with CC, based on the assessment of CTC and CD44+ CSC levels, was developed.

P53 Inhibits Bmi-1-driven Self-Renewal and Defines Salivary Gland Cancer Stemness.

Mucoepidermoid carcinoma (MEC) is a poorly understood salivary gland malignancy with limited therapeutic options. Cancer stem cells (CSC) are considered drivers of cancer progression by mediating tumor recurrence and metastasis. We have shown that clinically relevant small molecule inhibitors of MDM2-p53 interaction activate p53 signaling and reduce the fraction of CSC in MEC. Here we examined the functional role of p53 in the plasticity and self-renewal of MEC CSC. Using gene silencing and therapeutic activation of p53, we analyzed the cell-cycle profiles and apoptosis levels of CSCs in MEC cell lines (UM-HMC-1, -3A, -3B) via flow cytometry and looked at the effects on survival/self-renewal of the CSCs through sphere assays. We evaluated the effect of p53 on tumor development (N = 51) and disease recurrence (N = 17) using in vivo subcutaneous and orthotopic murine models of MEC. Recurrence was followed for 250 days after tumor resection. Although p53 activation does not induce MEC CSC apoptosis, it reduces stemness properties such as self-renewal by regulating Bmi-1 expression and driving CSC towards differentiation. In contrast, downregulation of p53 causes expansion of the CSC population while promoting tumor growth. Remarkably, therapeutic activation of p53 prevented CSC-mediated tumor recurrence in preclinical trials. Collectively, these results demonstrate that p53 defines the stemness of MEC and suggest that therapeutic activation of p53 might have clinical utility in patients with salivary gland MEC.

The Antiviral Drug Efavirenz in Breast Cancer Stem Cell Therapy.

Simple SummaryCancer stem cells (CSCs) are responsible for tumour initiation, chemo- and radiotherapy resistance and cancer recurrence. CSCs display plasticity that enables them to alter their phenotype and function making them challenging to eliminate. In this study we explore the effects of an antiretroviral medication used to treat HIV/AIDS (Efavirenz) on cancer stem cells derived from multiple breast cancer cell lines. Efavirenz has been previously found to be effective in the treatment of triple-negative breast cancers, and here we show that it is also capable of altering CSC numbers, cell morphology, RNA/microRNA gene expression and levels of epithelial/mesenchymal CSC subtypes. This study shows that, with Efavirenz, it is possible to not only eliminate primary breast cancer cells, but also to promote changes in cell morphology.Although many breast cancer therapies show initial success in the treatment of the primary tumour, they often fail to eliminate a sub-population of cells known as cancer stem cells (CSCs). These cells are recognised for their self-renewal properties and for their capacity for differentiation often leading to chemo/radio-resistance. The antiviral drug Efavirenz has been shown to be effective in eliminating triple-negative breast cancer cells, and here we examine its effect on breast CSCs. The effects of Efavirenz on CSCs for several breast cancer cell lines were investigated by examining cellular changes upon drug treatment, including CSC numbers, morphology, RNA/microRNA expression and levels of epithelial/mesenchymal CSC subtypes. Efavirenz treatment resulted in a decrease in the size and number of tumorspheres and a reduction in epithelial-type CSC levels, but an increase in mesenchymal-type CSCs. Efavirenz caused upregulation of several CSC-related genes as well as miR-21, a CSC marker and miR-182, a CSC suppressor gene. We conclude that Efavirenz alters the phenotype and expression of key genes in breast CSCs, which has important potential therapeutic implications.

261 Distinct Melanoma Cancer Stem Cell Profile Defines Response to Immune Checkpoint Inhibitors in Melanomas

This study examined the relationship between cancer stem cells (CSCs) and the susceptibility of melanomas to immunotherapy. Immunotherapy has emerged as a pillar of cancer therapy, demonstrating marked survival benefits in patients with melanoma. However, there remains a significant number of patients who fail to respond to treatment. To improve patient outcomes and optimise appropriate drug selection, it is a priority to unravel the mechanisms underlying the emergence of drug resistance. One of the most important key players to have emerged in the development of drug resistance are the CSCs. Transcriptomic profiles of 158 melanoma biopsies from patients treated with anti-programmed cell death 1 ligand-1 (PD-1) monotherapy (nivolumab or pembrolizumab; n = 63) or combined anti-PD-1 and anti-cytotoxic T lymphocyte antigen 4 (CTLA-4; ipilimumab; n = 57) were obtained. Patients were categorised as responders or non-responders using the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, where responders had a complete response (CR), partial response (PR), or stable disease (SD) of greater than 6 months with no disease progression. Samples were analysed for differential gene expression after normalisation using DESeq2 and HTSeq. Gene levels of well-defined CSCs were compared between responders and non-responders. Changes to gene expression levels were also compared pre and post immunotherapy treatment. This analysis identified a unique melanoma CSC marker gene expression profile “signature” that corresponds to a positive response to immune checkpoint inhibitors. This gene expression profile was associated with longer progression-free survival in patients treated with immunotherapy agents. Therefore, we have identified a CSC gene expression profile that can be used to predict patient response to the current available anti-PD-1 or anti-CTLA4 based therapies. Furthermore, this helps with identifying patients in need of other therapeutic strategies.

Chemotherapy-Enriched THBS2-Deficient Cancer Stem Cells Drive Hepatocarcinogenesis through Matrix Softness Induced Histone H3 Modifications.

The physical microenvironment is a critical mediator of tumor behavior. However, detailed biological and mechanistic insight is lacking. The present study reveals the role of chemotherapy‐enriched CD133+ liver cancer stem cells (CSCs) with THBS2 deficiency. This subpopulation of cells contributes to a more aggressive cancer and functional stemness phenotype in hepatocellular carcinoma (HCC) by remodeling the extracellular matrix (ECM) through the regulation of matrix metalloproteinase (MMP) activity, collagen degradation, and matrix stiffness. The local soft spots created by these liver CSCs can enhance stemness and drug resistance and provide a route of escape to facilitate HCC metastasis. Interestingly, a positive feed‐forward loop is identified where a local soft spot microenvironment in the HCC tumor is enriched with CD133 expressing cells that secrete markedly less ECM‐modifying THBS2 upon histone H3 modification at its promoter region, allowing the maintenance of a localized soft spot matrix. Clinically, THBS2 deficiency is also correlated with low HCC survival, where high levels of CSCs with low THBS2 expression in HCC are associated with decreased collagen fiber deposits and an invasive tumor front. The findings have implications for the treatment of cancer stemness and for the prevention of tumor outgrowth through disseminated tumor cells.

Abstract 5043: A vimentin binding small molecule (FiVe1) is a novel candidate to target CSC-rich TNBC

Abstract Introduction: Epithelial mesenchymal transition (EMT) and Cancer stem cells (CSCs) are associated with metastasis and tumor recurrence. We previously identified that EMT induces stem cell properties and the transcription factor FOXC2 is one of the key mediators. Recently, in a high throughput small molecule screen using FOXC2-overexpressing cells, we identified FiVe1 that binds to vimentin resulting in mitotic collapse and multinucleation. Vimentin is an intermediate filament that is upregulated during EMT and is present in CSCs, where it provides increased motility as well as resistance to mechanical stress in these cells. In this work, we hypothesize that FiVe1 selectively targets mesenchymal-looking, CSC-enriched populations via vimentin. Experimental procedures: Different subtypes of breast cancer cells and non-transformed cells, as well as CSC-enriched or depleted populations, were assessed for the occurrence of multinucleation (>2 nuclei) after FiVe1 treatment for two population doubling (2PD) through counting by fluorescent microscopy. Cell lines that became multinucleated were then tested for the reversibility of multinucleation after passaging cells with fresh media. Levels of vimentin phosphorylation (S56) and total vimentin were assessed by immunoblot. Summary of data: Multinucleation was observed only in the mesenchymal-like subtypes of TNBC cell lines relative to other TNBC subtypes and non-transformed cell lines. This multinucleation was irreversible upon passaging with fresh media. The levels of S56 phosphorylation of vimentin increased in these cell lines, but gradually decreased upon passaging with fresh media. To determine if multinucleation was due to the higher number of CSCs in mesenchymal-like TNBC cells, CSC-enriched populations were treated with FiVe1. The CSC-enriched populations had significantly increased levels of multinucleation upon FiVe1 treatment relative to CSC-depleted and control populations. Conclusions: The induction of multinucleation by Five1 correlates with higher CSC levels. This was observed both in the mesenchymal-like subtypes of TNBCs, which have higher stem-like properties relative to other TNBC subtypes, as well as cell populations enriched for CSCs. The increase in vimentin phosphorylation at S56 after FiVe1 treatment in TNBC cell lines is physiologically significant as misregulated S56 phosphorylation results in multinucleation and dysregulation of vimentin assembly. While the S56 phosphorylation decreased upon withdrawal of FiVe1 in all cells, the kinetics of reversibility is lower in mesenchymal-looking cells, which corroborate with the multinucleation. Further research regarding the fate of multinucleated cells is in progress. Inhibiting CSCs by targeting vimentin is a promising approach for sensitizing these inherently resistant tumors to chemotherapeutic agents. Citation Format: Nick A. Kuburich, Matthew Rosolen, Francisco Martinez, Petra den Hollander, Michael J. Bollong, Rama Soundararajan, Luke L. Lairson, Sendurai A. Mani. A vimentin binding small molecule (FiVe1) is a novel candidate to target CSC-rich TNBC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5043.

Predictors of recurrence and survival of hepatocellular carcinoma: A prospective study including transient elastography and cancer stem cell markers

Background and Study AimsTo investigate whether the measurement of liver stiffness (LSM) using fibroscan and the serum Cancer Stem Cells (CSC): Ep-CAM and cytokeratin-19, could predict the recurrence of hepatocellular carcinoma (HCC) and their impact on clinical outcome and overall survival. Patients and MethodsThis is a prospective study, including 179 HCV-related HCC patients. All patients were treated following the BCLC guidelines. All HCC patients had transient elastography, measurements of Ep-CAM and cytokeratin-19 before and six months post-treatment. We looked for predictors of recurrence and performed a survival analysis using Kaplan-Meier estimates. ResultsTACE was the most common procedure (77.1%), followed by microwave ablation (15.6%). Complete ablation was achieved in 97 patients; 55 of them developed HCC recurrence. After treatment, LSM increased significantly with a significant reduction in CSCs levels in complete and partial response groups. The median time to observe any recurrence was 14 months. LSM increased significantly post-treatment in patients with recurrence versus no recurrence. Higher levels of CSCs were recorded at baseline and post-treatment in patients with recurrence but without statistical significance. We used univariate analysis to predict the time of recurrence by determining baseline CK-19 and platelet levels as the key factors, while the multivariate analysis determined platelet count as a single factor. The univariate analysis for prediction of overall survival included several factors, LSM and EpCAM (baseline and post-ablation) among them, while multivariate analysis included factors such as Child score B and incomplete ablation. ConclusionDynamic changes were observed in LSM and CSCs levels in response to HCC treatment and tumour recurrence. Child score and complete ablation are factors that significantly affect survival.

Effect of Chemotherapeutic Agents on the Expression of Retinoid Receptors and Markers of Cancer Stem Cells and Epithelial-Mesenchymal Transition.

A large body of evidence suggests that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), as well as expression and function of retinoid receptors, are pivotal features of tumor initiation, progression, and chemoresistance. This is also true for pancreatic ductal adenocarcinoma (PDAC), which represents a clinical challenge due to poor prognosis and increasing incidence. Understanding the above features of cancer cells could open new avenues for PDAC treatment strategies. The aim of this study was to investigate the relation between CSCs, EMT, and retinoid receptors in PDAC after treatment with the chemotherapeutic agents - gemcitabine and 5-fluorouracil. First, we demonstrated the difference in the expression levels of CSC and EMT markers and retinoid receptors in the untreated Mia PaCa-2 and Panc1 cells that also differed in the frequency of spontaneous apoptosis and distribution between the cell cycle phases. Chemotherapy reduced the number of cancer cells in the S phase. Gemcitabine and 5-fluorouracil modulated expression of CSC markers, E-cadherin, and RXRβ in Panc1 but not in Mia PaCa-2 cells. We suggest that these effects could be attributed to the difference in the basal levels of expression of the investigated genes. The obtained data could be interesting in the context of future preclinical research.

Circulating hematopoietic stem cells, endothelial progenitor cells and cancer stem cells in hepatocellular carcinoma patients: contribution to diagnosis and prognosis

Background and aim: Circulating hematopoietic stem cells (HSCs), circulating endothelial progenitor cells (EPCs) and cancer stem cells (CSCs) contribute to tumor development and progression and can predict patient outcome. The aim of this study was to investigate the frequency of circulating HSCs, EPCs and CSCs in the peripheral blood of patients with hepatocellular carcinoma (HCC) and to explore their potential prognostic significance for HCC patients.Methods: The study included 30 HCC patients and 20 healthy controls. The HSCs and EPCs were enumerated with CD45, CD34, CD133, CD144 markers, while CSCs were enumerated with CD45, CD44, CD133 markers using flow cytometry.Results: The mean percentages of circulating HSCs were significantly lower in HCC patients than the controls (p = .001), whereas the mean percentages of EPCs within the HSCs subpopulation were significantly higher in the HCC patients than the controls (p = .002). The absolute count of CSCs within 100,000 peripheral blood mononuclear cells was 23.5 ± 3.4 in the HCC patients. Also, the mean percentages of circulating HSCs, EPCs and the number of CSCs were significantly increased in patients with multiple hepatic focal lesions than in patients with a single hepatic focal lesion. Both circulating HSCs and EPCs showed significant positive correlation with the level of AFP and with the numbers of CSCs. In the meantime, the numbers of CSCs revealed significant direct correlation with ALT, AST and AFP levels. The one-year overall survival (OS) of the patients was 77.5%. High levels of CSCs, HSCs and EPCs at diagnosis were all associated with worse outcome for the HCC patients.Conclusions: Significant changes in the levels of the circulating HSCs, EPCs and CSCs occur in HCC. These changes help the diagnosis and the prediction of HCC outcome, as higher levels of these cells are associated with worse OS.

Insluin and epithelial growth factor (EGF) promote programmed death ligand 1(PD-L1) production and transport in colon cancer stem cells

BackgroundProgrammed cell death ligand 1 (PD-L1) is an important immune-inhibitory protein expressed on cancer cells to mediate cancer escape through interaction with PD-1 expressed on activated T lymphocytes (T cells). Previously, we reported that colon and breast cancer stem cells (CSCs) expressed much higher levels of PD-L1 than their parental cells, suggesting they will be more resistant to immune attack.MethodsWe investigated the underlining mechanism of PD-L1 increase in colon CSCs, with a special focus on the effect of insulin and epithelial growth factor (EGF), the two fundamental components to sustain the metabolism and stemness in the culture of CSCs.ResultsWe found that insulin increased the total and surface PD-L1 levels through PI3K/Akt/mTOR pathway as the increase could be inhibited by the dual inhibitor of the pathway, BEZ235. EGF didn’t affect the total PD-L1 levels of CSCs but increased the cell surface protein levels by flow cytometry analysis, indicating EGF promotes the transport of PD-L1 to the cell surface. Blocking cell surface PD-L1 with a specific antibody resulted in a significant reduction of tumour sphere formation but didn’t interfere with the sphere growth, suggesting that cell surface PD-L1 may act as an adhering molecule for CSCs.ConclusionsApart from the essential roles in metabolism and stemness, insulin and EGF involve in up-regulation of PD-L1 expression in colon CSCs, therefore the inhibition of insulin and EGF/EGFR pathways can be considered for cancer immunotherapy or combined with PD-1/PD-L1 antibody-based cancer immunotherapy to eliminate CSCs.

B591, a novel specific pan-PI3K inhibitor, preferentially targets cancer stem cells

Cancer stem cells (CSCs) have been implicated in metastasis, relapse, and therapeutic resistance of cancer, so successful cancer therapy may therefore require the development of drugs against CSCs or combining anti-CSCs drugs with conventional therapies. The phosphoinositide 3-kinase (PI3K) signaling pathway is one of the most frequently activated signaling pathways in human cancer, playing a central role in tumorigenesis as well as the maintenance of CSCs. Here, we designed and identified B591, a dihydrobenzofuran-imidazolium salt, as a novel specific pan-PI3K inhibitor with potent inhibitory activity against class I PI3K isoforms, which showed effective inhibition of cellular PI3K/mTOR signaling pathway and robust antitumor activity in a set of cancer cell lines. Notably, compared with bulk tumor cell populations, B591 exhibited more potency in suppressing CSCs survival and inducing CSCs apoptosis, and presence of B591 effectively eliminated paclitaxel-enriched CSCs. B591 diminished self-renewal capacity and decreased the expression of epithelial-mesenchymal transition (EMT) markers of CSCs. In vivo, B591 preferentially decreased CSCs levels in mouse xenograft model of human breast cancer as evidenced especially by remarkable reduction of tumor-initiating ability. Consistent with the preferential targeting of CSCs, B591 effectively inhibited breast tumor metastasis and delayed tumor regrowth following paclitaxel treatment. Taken together, our findings establish B591, a novel PI3K inhibitor, as a strong candidate for clinical evaluation as a CSCs targeting agent.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Correlation of 18F-FDG uptake and thyroid cancer stem cells.

2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography (18F-FDG PET) has the potential to detect various types of cancers, including thyroid cancer (TC), at a potentially curable stage. Increased uptake of 18F-FDG was observed in anaplastic and poorly differentiated thyroid cancer cells, and PET-positive tumors are more likely to be resistant to 131I treatment. As cancer stem cells (CSCs) possess a dedifferentiated phenotype and are resistant to many anticancer therapies, we hypothesized that the expression of CSC-related markers is correlated with the ability of tumor cells in TC to uptake FDG. The present study cohort included 12 patients with TC, who underwent 18F-FDG PET/CT imaging before surgery. Quantitative polymerase chain reaction (QPCR) and immunohistochemical (IHC) staining were performed to analyze the expression patterns of gene markers related to embryonic stem (ES) cells and CSCs in TC. The mRNA expression levels of CSC- (CD133 and CD44) and ES-related genes (Oct4 and Nanog) were higher in TC tissue than in normal thyroid tissue, whereas the mRNA expression levels of thyroid-specific genes (Tg, TSHR, and TTF1) were higher in normal thyroid tissue than in TC tissue. There was a positive and statistically significant correlation between FDG uptake (SUV<inf>max</inf>) of tumor and relative mRNA levels of CD133, CD44, Oct4, and Nanog. The IHC results demonstrated that CD133 and Nanog were expressed in TC tissue but not in normal thyroid tissue, however, CD44 expression was observed in both TC and normal thyroid tissue. Comparisons of the clinicopathological parameters between TC tissues with low and high SUV<inf>max</inf> demonstrated significant differences in protein level of CD133 but not in that of Nanog. The pre-therapeutic tumor SUV<inf>max</inf> obtained from 18F-FDG PET/CT may be a potential predictor for evaluating the proportion of CSC population in individual patients with TC.

Cancer stem cells in colorectal tumors of various extents.

e15659Background: The purpose of the study was to assess an association between the tumor invasion and levels of cancer stem cells (CD44+ and CD133+) in colorectal cancer. Methods: The study includ...

MiR-4319 Suppress the Malignancy of Triple-Negative Breast Cancer by Regulating Self-Renewal and Tumorigenesis of Stem Cells

Background/Aims: High levels of cancer stem cells (CSCs) in patients with triple-negative breast cancer (TNBC) correlate with risk of poor clinical outcome and possibly contribute to chemoresistance and metastasis in patients with highly malignant TNBC. Aberrant microRNA expression is associated with the dysfunction of self-renewal and proliferation in cancer stem cells, while there is little information about the TNBC-specific microRNAs in regulating CSC ability. Methods: Solexa deep sequencing was performed to detect the expression levels of TNBC or non-TNBC stem cells (CSCs) microRNAs. Mammosphere formation assay, qRT-PCR and the xenograft model in nude mice were performed. Bioinformatic analysis and microarray were used to select the target gene, and luciferase reporter assays were used to confirm the binding sites. Results: Solexa sequencing data exhibited differential expression of 193 microRNAs between TNBC and non-TNBC stem cells. The gene ontology analysis and pathways analyses showed that genes were involved in the maintenance of stemness. MiR-4319 could suppress the self-renewal and formation of tumorspheres in TNBC CSCs through E2F2, and also inhibited tumor initiation and metastasis in vivo. Moreover, increased E2F2 could reverse the effect of miR-4319 on the self-renewal in TNBC CSCs. Conclusions: MiR-4319 suppresses the malignancy of TNBC by regulating self-renewal and tumorigenesis of stem cells and might be a remarkable prognostic factor or therapeutic target for patients with TNBC.