Stem Cell Marker CD133 Research Articles

Therapeutic targeting of hepatocellular carcinoma cells with antrocinol, a novel, dual-specificity, small-molecule inhibitor of the KRAS and ERK oncogenic signaling pathways

Until recently, sorafenib has been the only treatment approved by the U.S. Food and Drug Administration for patients with advanced hepatocellular carcinoma (HCC). Some patients, however, exhibit resistance to this treatment and subsequently experience cancer progression, recurrence, or death. Therefore, identifying a new alternative treatment for patients with little or no response to sorafenib treatment is vital. In this study, we explored the therapeutic potential and underlying molecular mechanism of antrocinol ((3aS,4R,6aS,10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphtho[1,8a-c]furan-1-one) in patients with HCC. The results indicated that antrocinol was more therapeutically effective than antrocin, Stivarga, and sorafenib against HCC cell lines. Antrocinol also substantially suppressed the expression of KRAS-GTP, p-MEK1/2, p-ERK1/2, and p-AKT in the Huh7 cell line. Additionally, antrocinol-induced apoptosis in the Huh7 cell line, inhibited the formation of tumorspheres, and suppressed the expression of cancer stem cell markers CD133, KLF4, CD44, OCT4, SOX2, and c-Myc. Animal studies revealed that antrocinol alone considerably suppressed tumor growth in nonobese diabetic/severe combined immunodeficient mice inoculated with Huh7 tumorspheres. It also synergistically enhanced the anticancer effect of sorafenib, resulting in enhanced suppression of tumor growth (p < 0.001) and tumorsphere formation (p < 0.001). In tumor samples resected from mice treated with antrocinol alone or in combination with sorafenib, immunohistochemical analysis revealed an increase in BAX expression and a decrease in ERK and AKT protein expression. To the best of our knowledge, this is the first report of the anti-HCC activity of antrocinol. With its higher therapeutic efficacy than that of sorafenib, antrocinol is a candidate drug for patients with HCC who demonstrate little or no response to sorafenib treatment.

Association of cancer stem cell markers CD44 and CD133 expression with clinicopathological changes in Oral squamous cell carcinoma (OSCC) and oral submucosal fibrosis (OSMF)

Oral squamous cell carcinoma (OSCC) is a major health problem worldwide. Recently, cancer stem cell (CSC) biomarkers were examined due to their impact on tumorigenesis and recurrence. In the present study, we aimed to examine the expression of CSC markers: CD133 and CD44 in tobacco-induced OSCC and oral submucosal fibrosis (OSMF) patients and we simultaneously evaluated their association with clinicopathological changes. A total of 60 cases (OSCC) and 60 controls (OSMF) were enrolled in the study with tobacco-based habits. Tumor appearance grade wise was characterized in OSMF and OSCC groups. Clinicopathological parameters were analyzed in OSCC cases only. The extent of positive staining was scored 0-4. The results indicated that the tobacco intake habits (chewing, smoking or both) have significant effects on OSCC and OSMF changes. In maximum subjects (29) of OSCC cases, tumour appearance was in grade III with no such change in OSMF. IHC results showed 83 % subjects were positive for CD44 and CD133 in OSCC cases, while 72 % were positive for CD44 and 76% for CD133 in the OSMF group. In the OSCC group the significant clinicopathological changes were observed such as tumour size in the range of 3-4 cm, T-3 staging, N-1 node involvement, enhanced metastasis and well differentiated pathological grade. In the OSCC group, high IHC score of 3 and 4 were observed in 81% of CD133 and 80.4% of CD44 positive subjects with tumor size of 3 to 4 cm, 74% of CD133 and 84% of CD44 positive individuals in the N-1 stage, 81.2% of CD133 and 85.7% of CD44 positive subjects at T-3 stage and 80.4% of CD133 and 66.6% of CD44 positive individuals with enhanced metastasis. These results clearly indicated clinicopathological changes in the OSCC group with parallel high expression of CD44 and CD133, these results showed the diagnostic relevance of CSC markers in the OSCC and OSMF subjects.

Loss of Epidermal Homeostasis Underlies the Development of Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is one of the most common skin cancers. To develop targeted therapies for SCC, a comprehensive understanding of the disease through a systems approach is required. Here, we have collated and analyzed the literature on SCC and pathways that maintain skin homeostasis. Since, the loss of the Notch and the overactivation of the Wnt pathways in the epidermis cause SCC, we focused on these two pathways. We found that the two pathways are critical in maintaining epidermal homeostasis. Further, we found that the cancer stem cell (CSC) marker CD44 causes the transcription of SOX2, another CSC marker of SCC, activates the Wnt pathway, and blocks the Notch pathway. Similarly, the Wnt pathway causes the transcription of CD44 and SOX2 and blocks the Notch pathway. In this paper, we have discussed how the notch and the Wnt pathways affect epidermal homeostasis and the three CSCs (CD44, SOX2, and LGR6) affect the two pathways, linking the CSCs with epidermal homeostasis.

Reliability of CD44, CD24, and ALDH1A1 immunohistochemical staining: Pathologist assessment compared to quantitative image analysis.

The data on the expression of stem cell markers CD44, CD24, and ALDH1A1 in the breast tissue of cancer-free women is very limited and no previous studies have explored the agreement between pathologist and computational assessments of these markers. We compared the immunohistochemical (IHC) expression assessment for CD44, CD24, and ALDH1A1 by an expert pathologist with the automated image analysis results and assessed the homogeneity of the markers across multiple cores pertaining to each woman. We included 81 cancer-free women (399 cores) with biopsy-confirmed benign breast disease in the Nurses' Health Study (NHS) and NHSII cohorts. IHC was conducted with commercial antibodies [CD44 (Dako, Santa Clara, CA, USA) 1:25 dilution; CD24 (Invitrogen, Waltham, MA, USA) 1:200 dilution and ALDH1A1 (Abcam, Cambridge, United Kingdom) 1:300 dilution]. For each core, the percent positivity was quantified by the pathologist and Definiens Tissue Studio. Correlations between pathologist and computational scores were evaluated with Spearman correlation (for categorical positivity: 0, >0-<1, 1-10, >10-50, and >50%) and sensitivity/specificity (for binary positivity defined with 1 and 10% cut-offs), using the pathologist scores as the gold standard. Expression homogeneity was examined with intra-class correlation (ICC). Analyses were stratified by core [normal terminal duct-lobular units (TDLUs), benign lesions] and tissue type (epithelium, stroma). Spearman correlation between pathologist and Definiens ranged between 0.40-0.64 for stroma and 0.66-0.68 for epithelium in normal TDLUs cores and between 0.24-0.60 for stroma and 0.61-0.64 for epithelium in benign lesions. For stroma, sensitivity and specificity ranged between 0.92-0.95 and 0.24-0.60, respectively, with 1% cut-off and between 0.43-0.88 and 0.73-0.85, respectively, with 10% cut-off. For epithelium, 10% cut-off resulted in better estimates for both sensitivity and specificity. ICC between the cores was strongest for CD44 for both stroma and epithelium in normal TDLUs cores and benign lesions (range 0.74-0.80). ICC for CD24 and ALDH1A ranged between 0.42-0.63 and 0.44-0.55, respectively. Our findings show that computational assessments for CD44, CD24, and ALDH1A1 exhibit variable correlations with manual assessment. These findings support the use of computational platforms for IHC evaluation of stem cell markers in large-scale epidemiologic studies. Pilot studies maybe also needed to determine appropriate cut-offs for defining staining positivity.

Tumor-associated macrophages promote resistance of hepatocellular carcinoma cells against sorafenib by activating CXCR2 signaling

BackgroundSorafenib (SOR) is the first line treatment for advanced hepatocellular carcinoma (HCC), but resistance develops frequently. Tumor-associated macrophages (TAMs) have been reported to affect the progression of HCC. We therefore aimed to study the role of TAMs in promoting SOR resistance.MethodsImmunofluorescence staining for the M2 marker CD204 and the cancer stem cell (CSC) markers CD44 and CD133 was performed in paired HCC and adjacent noncancerous tissues and HCC tissues stratified by response of SOR treatment. HCC/U937 coculture system and cytokines were used to induce M2 polarization for studying the effects of M2 TAMs on CSC properties and apoptotic death of HCC cells after SOR treatment.ResultsHigher expression of CD204, CD44, and CD133 was observed in patients with SOR nonresponse (SNR) than in those with SOR response (SR), suggesting that SNR is positively correlated to levels of CSCs and M2 TAMs. After coculture, M2 TAMs could increase the level of CSCs but decrease SOR-induced apoptosis. Incubation of HCC cells with coculture conditioned medium increased the formation of spheres that were resistant to SOR. Furthermore, CXCL1 and CXCL2 were found to be the potential paracrine factors released by M2 TAMs to upregulate SOR resistance in HCC cells. Treatment with CXCL1 and CXCL2 could increase HCC CSC activity but decrease SOR-induced apoptosis by affecting BCL-2 family gene expression. Using pharmacological inhibitors, CXCR2/ERK signaling was found to be critical to CXCL1- and CXCL2-mediated SOR resistance.ConclusionThis study identified CXCL1, CXCL2, and their downstream CXCR2/ERK signaling as potential therapeutic targets to overcome SOR resistance in HCC.

Bone Marrow Plasma Proteome Analysis By Differential Ultracentrifugation and Tandem Mass Spectrometry Allow for Characterization of Leukemia-Derived Extracellular Vesicles in Patients with Acute Lymphoblastic Leukemia

In recent years, the interplay of leukemic cells with the bone marrow niche has emerged as a potential contributing factor for the survival, therapy resistance and relapse-initiating capacity of leukemic cell subsets in ALL. This is known as early, environment-mediated drug resistance (EMDR). The role for leukemia-derived extracellular vesicles (LEVs) in this interplay remains largely unexplored. In vitro studies and PDX models have demonstrated that LEVs are internalized by bone marrow stromal cells (BMSC), leading to BMSC reprogramming and metabolic switch to glycolysis, with beneficial effects on leukemic cell survival as a result. This suggests that LEVs could also play a role in leukemic cell survival in ALL during chemotherapy, however, studies examining LEVs in patients are warranted. We performed fractionation by differential ultracentrifugation and subsequent mass spectrometry analysis of bone marrow plasma samples (n=12) collected at time of diagnosis (n=6, one sample was processed at 30, 60 and 120 min after sampling to assess the potential effect of contamination with platelet-derived EVs), during (n=2) and after induction therapy (n=4) in patients with B-cell precursor ALL (n=4). We isolated four fractions: A (2,000g pellet): Enriched in platelets/apoptotic bodies, M (20,000g pellet, enriched in microvesicles: large, membrane-derived EVs), E (150,000g pellet, enriched in exosomes), and S (150,000g supernatant, mostly soluble proteins), as well as R (raw untreated plasma), and we subjected these to mass spectrometry-based quantitative proteomics in 3 technical replicates. We examined protein composition and enriched pathways in the fractions and investigated associations of EV protein levels with the size of the leukemic cell fraction in the bone marrow. Flowcytometric data was used to confirm cell distributions/surface marker expression. Examining the protein composition of the five fractions (A, M, E, S, and R) showed that although the highest number of proteins was found in the A fraction (2803 proteins), the M fraction (2477 proteins) had the highest proteome diversity (Figure 1). Focusing on the M fraction, we compared diagnostic with follow up samples (blast percentage day 0: 92, 95, and 72, day 15: 0.4 and 1.3, day 29: 0.05, 0.2, 0, and 0.001, one day 0 sample excluded due to poor readout quality). We found significant upregulation of B-cell associated proteins at diagnosis, incl. HLA-DR, CD19, and CD22, along with the stem cell marker CD34, while T-cell and platelet-associated proteins did not show this pattern. Flow cytometry demonstrated only very few healthy B-cells in the samples at time of diagnosis (5, 0.8, and 0.6% vs. 25, 3, 32 and 21% on day 29) and showed all the leukemic clones to be CD34positive, confirming the BCP-ALL leukemic origin of the EVs. Examining the proteins most closely correlated to the percentage of blasts across all the samples, we found several proteins associated with poor survival, cell migration/adhesion and chemoresistance in acute leukemia, including CD97 (r=0.85, p=0.006), PLXNB2 (r=0.72, P=0.037), and ITGA4 (r=0.6, p=0.097). Gene set enrichment analysis of the proteins showing the most prominent differences between day 0 and day 15/29 using Reactome identified a variety of metabolic processes, but also oncogenic MAPK signaling (NES 1.94, p-adj=0.005), integrin signaling (NES 1.94, p-adj=0.003), and regulation of expression of SLITs and ROBOs (NES 2.1, p-adj=3x10-7) as enriched among the day 0 samples. Interestingly, we recently found genes within the latter two selectively upregulated in leukemias with poor induction therapy response (Modvig et al, Molecular Oncology, 2022). Taken together, this suggests that LEVs indeed could play a role in leukemic cell adherence and EMDR in the bone marrow niche. In conclusion, LEV content can be reliably characterized in bone marrow plasma of patients with ALL through differential ultracentrifugation and mass spectrometry-based proteomics. LEV protein composition reflects processes such as leukemic adhesion/migration, survival and drug resistance, carefully suggesting a role for LEVs in EMDR, but larger studies in patients are warranted. The identification of LEVs and key pathways in the leukemic-stromal cell interplay/EMDR can lead to development of new drugs blocking this cross-talk, potentially contributing to an improved relapse-free survival in patients with ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Establishment and Characterization of a Model of Acquired Resistance to DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic Leukemia Cells

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) is an aggressive malignancy with event-free survival rates of only ~37%. KMT2A translocation represents the cytogenetic hallmark of this type of leukemia that is largely driven by inappropriate epigenetic events: oncogenic KMT2A fusion proteins recruit the histone methyltransferase DOT1L which leads to misplaced H3K79 methylation inducing an abnormal transcriptomic landscape that favors leukemia development. Therefore, the development and subsequent (pre-)clinical testing of the specific DOT1L inhibitor pinometostat was anticipated to be key to the successful treatment of KMT2A-rearranged acute leukemia. However, although clinical trials in adult KMT2A-rearranged acute leukemia patients did report initial responses (including complete remissions), disease progression due to treatment-related resistance appeared unavoidable. Nonetheless, while these observations urge the development of more effective inhibitors of DOT1L, the mechanisms underlying acquired resistance to DOT1L inhibition may well provide valuable insights into the unique biology of KMT2A-rearranged leukemia. To explore this, we provoked and established acquired pinometostat resistance in a pediatric KMT2A::AFF1+ ALL cell line model that stably became ~35-fold more resistant to DOT1L inhibition. Interestingly, pinometostat was still able to completely inhibit H3K79 di-methylation (H3K79me2) but failed to induce apoptosis, indicating that these cells largely became independent of DOT1L-mediated H3K79 methylation. Yet, shRNA-mediated knock-down of KMT2A::AFF1 revealed that these cells remained completely dependent on the presence of the KMT2A-fusion protein. In addition, pinometostat-resistant KMT2A-rearranged ALL cells proliferate more slowly and appeared to have switched their preference from oxidative phosphorylation to glycolysis to provide energy for metabolic activities, while remaining perfectly viable. As anticipated, pinometostat-resistant cells displayed a slight cross-resistance to most chemotherapeutic agents currently used in ALL treatments but became remarkably more sensitive toward the BCL-2 inhibitor venetoclax. To further characterize these cells and gain insights into the underlying mechanisms of resistance, we performed RNA-, ATAC-, and ChIP-sequencing analysis of KMT2A and AFF1 as well as of histone marks essential in KMT2A-rearranged ALL, including H3K79me2, H3K4 tri-methylation (H3K4me3) and H3K27 acetylation (H3K27ac). This revealed that acquired resistance to DOT1L inhibition leads to a selective loss of KMT2A-fusion driven epigenetic regulation and expression of genes such as PROM1 (encoding the hematopoietic/ leukemia stem cell marker CD133) and its enhancer TAPT1, as well as putative KMT2A::AFF1 target genes including RUNX2, GNAQ, SERPINB1, PRSS12, and ZC3H12C. In contrast, however, the expression of (and levels of H3K79 methylation at) other established KMT2A::AFF1 target genes like HOXA9, HOXA10, MEIS1, and CDK6 remained unaffected. Concomitantly, pinometostat-resistant KMT2A-rearranged ALL cells are characterized by significant enrichment of gene expression of various MYC targets, and specific up-regulation of genes associated with a myeloid immunophenotype, including CD33, CD38, LILRB4, MPEG1, LIMK1, and CCL5. Taken together, we here present an in vitro model of acquired resistance to DOT1L inhibition in KMT2A-rearranged ALL, instantly providing new insights and raising important questions regarding this elusive type of leukemia. For instance, KMT2A-rearranged ALL cells readily adapt to prolonged inhibition of H3K79 methylation but remain dependent on the KMT2A fusion, suggesting that recruitment of DOT1L may not be the only oncogenic action exerted by KMT2A fusion proteins. Also, our model demonstrates that under prolonged pressure of DOT1L inhibition, KMT2A-rearranged ALL cells seem to initiate a reprogramming process that involves the acquirement (or selection) of myeloid-like characteristics, an ability that may be connected to leukemic lineage switches which are not uncommon in KMT2A-rearranged acute leukemias. Hence, our model represents an important tool to study the complex biology of KMT2A-rearranged leukemia, and its existence and availability requires to be shared with the community.

CD44 is highly expressed in stem/progenitor cells originating the intervertebral discs in the human notochord.

The notochord acts as a patterning structure, playing a key role in the formation of the vertebral column, both indirectly by inducing sclerotome cell differentiation and directly by forming the nucleus pulposus of intervertebral discs. The abnormal development of the notochord results in an easy equation with a variety of birth defects. Therefore, we focused our attention on the analysis of the early stages of human notochord development by highlighting the role of progenitor stem cells involved in the origin of intervertebral discs (IVDs). Eight human fetuses, ranging from 8 up to 21 weeks of gestational age, were obtained from spontaneous abortion or voluntary interruption of gestation. Samples were 10% formalin-fixed, routinely processed, and paraffin-embedded. Five micron-tick paraffin sections were obtained from each sample. Sections were stained with hematoxylin-eosin and PAS stain for a morphological examination. Tissue samples were immunostained with a commercial anti-human CD44 rabbit monoclonal antibody at 1:100 dilution. Immunoreactivity for CD44 was detected in six out of eight notochords examined in this study. Reactivity for CD44 was restricted to progenitor cells giving rise to the nucleus pulposus (NP) of the developing IVDs. Positive cells showed a membranous and/or cytoplasmic immunostaining, no reactivity was observed in the nuclear compartment. CD44 expression was always restricted to IVD precursor cells, whereas cartilage precursors were devoid of labelling. Our study shows, for the first time, that the stem cell marker CD44 selectively marks intervertebral disc progenitor cells, paralleling their differentiation toward a discogenic phenotype. Therefore, our results suggest that CD44 plays a key role in IVD development, allowing its differentiation from surrounding undifferentiated notochordal cells toward a IVD phenotype. Given the role of CD44 in IVD development, we may hypothesize that low CD44 levels might be associated with changes in IVD development and with susceptibility to develop back pain later in life.

HIV-1 Proteins gp120 and Tat Promote Epithelial-Mesenchymal Transition and Invasiveness of HPV-Positive and HPV-Negative Neoplastic Genital and Oral Epithelial Cells.

The incidence of human papillomavirus (HPV)-associated anogenital and oropharyngeal cancer in human immunodeficiency virus (HIV)-infected individuals is substantially higher than in HIV-uninfected individuals. HIV may also be a risk factor for the development of HPV-negative head and neck, liver, lung, and kidney cancer. However, the molecular mechanisms underlying HIV-1-associated increase of epithelial malignancies are not fully understood. Here, we showed that HPV-16-immortalized anal AKC-2 and cervical CaSki epithelial cells that undergo prolonged exposure to cell-free HIV-1 virions or HIV-1 viral proteins gp120 and tat respond with the epithelial-mesenchymal transition (EMT) and increased invasiveness. Similar responses were observed in HPV-16-infected SCC-47 and HPV-16-negative HSC-3 oral epithelial cancer cells that were cultured with these viral proteins. EMT induced by gp120 and tat led to detachment of poorly adherent cells from the culture substratum; these cells remained capable of reattachment, upon which they coexpressed both E-cadherin and vimentin, indicative of an intermediate stage of EMT. The reattached cells also expressed stem cell markers CD133 and CD44, which may play a critical role in cancer cell invasion and metastasis. Inhibition of transforming growth factor (TGF)-β1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasive activity of HPV-16-immortalized anal and cervical epithelial cells. Collectively, our results suggest that these approaches along with HIV viral suppression with antiretroviral therapy (ART) might be useful to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia. IMPORTANCE HPV-16-immortalized genital and oral epithelial cells and HPV-negative oral cancer cells that undergo prolonged contact with cell-free HIV-1 virions or with viral proteins gp120 and tat respond by becoming more invasive. EMT cells induced by HIV-1 in cultures of HPV-16-immortalized anal and cervical epithelial cells express the stem cell markers CD133 and CD44. These results suggest that the interaction of HIV-1 with neoplastic epithelial cells may lead to their de-differentiation into cancer stem cells that are resistant to apoptosis and anti-cancer drugs. Thus, this pathway may play a critical role in the development of invasive cancer. Inhibition of TGF-β1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasiveness of HPV-16-immortalized anal and cervical epithelial cells. Taken together, these results suggest that these approaches might be exploited to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia.

Microfluidic chip with reversible interface for noninvasive remission status monitoring and prognosis prediction of acute myeloid leukemia

Acute myeloid leukemia (AML) requires close monitoring of remission status for timely disease management. Liquid biopsy serves as a noninvasive approach for evaluating treatment response and guiding therapeutic modifications. Herein, we designed a non-invasive Leukemic stem cell Specific Capture Chip (LSC-Chip) with reversible recognition interface for AML remission status monitoring and prognosis prediction. A stem cell marker CD34 antibody coated herringbone chip with disulfide linkers was designed to capture and release leukemic stem cells (LSCs) in peripheral blood for efficient LSC enumeration and downstream single-cell analysis. Samples from 32 AML patients and 10 healthy donors were recruited for LSC enumeration and prognosis-associated subtyping with panels of official LSC markers (CD34+/CD123+/CD38-) and (Tie2+/CD34+/CD123+/CD38-), respectively. A cutoff value of 2.5 LSCs per milliliters of peripheral blood can be used to precisely distinguish non-remission AML patients from complete remission group. Moreover, single-cell RNA-seq of LSCs was performed to check different transcriptional profiles of LSC subtypes. Overall, the LSC-Chip with reversible recognition interface enabled reliable detection of LSCs from AML patient samples for noninvasive remission status monitoring and prognosis prediction in clinical AML management.

Evaluation the role of CD34 in patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common leukemia among the adult population and accounts for about 80% of all cases, and its characterized by clonal expansion of immature “blast cells” in the peripheral blood and bone marrow. The stem cell marker CD34 is expressed by leukemia blasts only for a subset of patients with acute myelogenous leukemia (AML). The aim of this study is to determine the role of CD34 in AML and its relationship with the parameters of complete blood (CBC). A total of (60) venous blood sample from patients and (30) control their ages ranged between 18 and 66 years, the samples was collected from Baghdad medical city hospital. Sera from all participants were tested to assessment CD34 level by ELISA. In the current study, CD34 in patients and control groups show a significant difference in relation with AML patients at p-value (&lt;0.001). The levels of CD34 in the patients were 0.55 ± 0.03, whereas it's significantly increased to 0.81 ± 0.05 in the control. Also we found the majority of patients presented with subtype M3 38.3% of total patients.

Evaluation the role of CD34 in patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common leukemia among the adult population and accounts for about 80% of all cases, and its characterized by clonal expansion of immature “blast cells” in the peripheral blood and bone marrow .The stem cell marker CD34 is expressed by leukemia blasts only for a subset of patients with acute myelogenous leukemia (AML).The aim of this study is to determine the role of CD34 in AML and its relationship with the parameters of complete blood (CBC). A total of (60) venous blood sample from patients and (30) control their ages ranged between 18 and 66 years, The samples was collected from Baghdad medical city hospital. Sera from all participants were tested to assessment CD34 level by ELISA.In the current study, CD34 in patients and control groups show a significant difference in relation with AML patients at p-value (&lt; 0.001). The levels of CD34 in the patients were 0.55 ± 0.03, whereas it’s significantly increased to 0.81 ± 0.05 in the control. also we found The majority of patients presented with subtype M3 38.3% of total patients.

Over Expression of Cancer Stem Cell Marker CD44 and Its Clinical Significance in Patients with Oral Squamous Cell Carcinoma.

Cancer stem cell marker CD44 is a cell-surface glycoprotein which is involved in various cellular functions such as cell-cell interactions, cell adhesion, haematopoiesis and tumour metastasis. The CD44 gene transcription is partly activated by beta-catenin and Wnt signalling pathway, the later pathway being linked to tumour development. However, the role of CD44 in oral squamous cell carcinoma (OSCC) is not well understood. We investigated the expression of CD44 in peripheral circulation, tumour tissues of oral cancer patients and oral squamous cell carcinoma cell lines by ELISA and quantitative (q)-RTPCR. Relative CD44s mRNA expression was significantly higher in peripheral circulation (p = 0.04), tumour tissues (p = 0.049) and in oral cancer cell lines (SCC4, SCC25 p = 0.02, SCC9 p = 0.03). Circulating CD44total protein levels were also significantly (p < 0.001) higher in OSCC patients that positively correlated with increasing tumour load and loco-regional spread of the tumour. The circulating tumour stem cell marker CD44 appears to be a potent indicator of tumour progression and may be useful for developing suitable therapeutics strategies for patients with oral squamous cell carcinoma.

EID3 Promotes Cancer Stem Cell-Like Phenotypes in Osteosarcoma through the Activation of PI3K-AKT Signaling Pathway.

The aim of this study is to elucidate molecular mechanism by which E1A-like inhibitor of differentiation 3 (EID3) promotes cancer stem cell-like phenotypes in osteosarcoma. Overexpression of EID3 in osteosarcoma cells generated more spherical clones, enhanced the expression of stemness-associated genes, and promoted chemoresistance, invasion, and metastasis. Furthermore, osteosarcoma cells overexpressing EID3 had increased ability to grow in suspension as osteospheres with high expression of Sox2 and stem cell marker CD133. In addition, knockdown of EID3 reduced sphere formation and inhibited osteosarcoma cell migration and invasion. RNA sequencing and bioinformatics analysis revealed that PI3K-Akt signaling pathway and MAPK pathway­related genes were enriched in osteosarcoma cells with high expression of EID3. Taken together, EID3 promotes osteosarcoma, and EID3–PI3K-Akt axis is a potential therapeutic target for osteosarcoma treatment.

Expression and Prognostic Significance of Stem Cell Marker CD133 in Survival Rate of Patients with Colon Cancer.

Today, colon cancer is one of the most common types of gastrointestinal cancer worldwide. CD133 as a known cancer stem cell marker has been found effective in cell proliferation and differentiation in various cancers, including colon cancer. We aimed to investigate the relationship between CD133 expression in colon cancer with prognostic factors and survival rate of patients with colon cancer by immunohistochemistry. Formalin-fixed paraffin-embedded (FFPE) tissue was taken from patients with colon cancer. Histopathology examination was done using hematoxylin and eosin staining. Immunohistochemistry was performed to determine CD133 expression. Association between CD133 expression and clinicopathological profile was then assessed. There was a statistically significant association between CD133 protein expression and sex , cancer stage, and lymphatic invasion (p = 0.044, p = 0.131, and p = 0.002, respectively). However, no significant correlation was identified between CD133 expression and other factors, including age of patients with colorectal cancer (CRC) (p= 0.267), tumor location (p= 0.494), tumor differentiation grade (p = 0.263), neural tissue invasion, and 5-year survival (p = 0.054). CD133 is a useful predictive or prognostic biomarker for CRC in clinical assessment and may serve as a potential therapeutic target for CRC.

CHOLESTEROL DEPLETION SUPPORT A STEM CELL–LIKE AND PROLIFERATIVE PROFILE IN OSCC CELL LINE

Objectives Oral squamous cell carcinoma (OSCC) is able to invade and develop metastasis. Cancer stem cells have been associated with tumor development, progression, and recurrence of OSCC. Tumor cells have high cholesterol rates in their membranes, and cholesterol depletion affects these cells and interferes in carcinogenesis. The objective was to evaluate the effect of cholesterol depletion with methyl-β-cyclodextrin (MβCD) on the expression of stem cell markers CD44 and ESA in OSCC cell line SCC-9. Study Design SCC-9 cells were depleted with MβCD at concentrations 0, 7.5, 10, and 15 mM and evaluated by flow cytometry for CD44 and ESA markers. Results An increase in the CD44-high/ESA-high population was observed after cholesterol depletion at all evaluated concentrations. This increase was accompanied by a decrease in the CD44-low population, indicating that depletion favors the selection of stem cell—like cells. There was no difference in the percentage of CD44-high/ESA-low cells in the treated groups compared to the control group. Conclusions Cholesterol depletion seems to induce a stem cell–like profile with greater proliferative capacity in OSCC cells. Oral squamous cell carcinoma (OSCC) is able to invade and develop metastasis. Cancer stem cells have been associated with tumor development, progression, and recurrence of OSCC. Tumor cells have high cholesterol rates in their membranes, and cholesterol depletion affects these cells and interferes in carcinogenesis. The objective was to evaluate the effect of cholesterol depletion with methyl-β-cyclodextrin (MβCD) on the expression of stem cell markers CD44 and ESA in OSCC cell line SCC-9. SCC-9 cells were depleted with MβCD at concentrations 0, 7.5, 10, and 15 mM and evaluated by flow cytometry for CD44 and ESA markers. An increase in the CD44-high/ESA-high population was observed after cholesterol depletion at all evaluated concentrations. This increase was accompanied by a decrease in the CD44-low population, indicating that depletion favors the selection of stem cell—like cells. There was no difference in the percentage of CD44-high/ESA-low cells in the treated groups compared to the control group. Cholesterol depletion seems to induce a stem cell–like profile with greater proliferative capacity in OSCC cells.

Comparison of Sources and Methods for the Isolation of Equine Adipose Tissue-Derived Stromal/Stem Cells and Preliminary Results on Their Reaction to Incubation with 5-Azacytidine.

Simple SummaryThe function of the equine heart is different from that in other species, and a species-specific in vitro model would simplify investigations in the field of equine cardiology. The recent advances in stem cell research and the availability of adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point for the development of such an in vitro model. In order to test the hypothesis that equine ASCs can be differentiated into cells resembling heart cells, we isolated ASCs from abdominal, retrobulbar, and subcutaneous adipose tissue after collagenase digestion or from direct cultivation of explants. Both techniques resulted in similar yields of cells displaying morphological, immunophenotypical, and molecular biological characteristics of mesenchymal stem cells. Abdominal adipose tissue was found to be most suitable for ASC isolation in equines. However, contrasting earlier studies performed with ASCs from other species, equine ASCs were refractory to 5-azacytidine-induced upregulation of markers characteristic for the differentiation into heart cells. Hence, further studies are required to establish equine cardiomyocyte induction.Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASCs and both isolation methods resulted in comparable yields of CD45-/CD34-negative cells expressing the mesenchymal stem cell markers CD29, CD44, and CD90, as well as pluripotency markers, as determined by flow cytometry and real-time quantitative PCR. However, exposure of equine ASCs to 5-azacytidine (5-AZA), reportedly inducing CM differentiation from rats, rabbits, and human ASCs, was not successful in our study. More precisely, neither the early differentiation markers GATA4 and NKX2-5, nor the late CM differentiation markers TNNI3, MYH6, and MYH7 were upregulated in equine ASCs exposed to 10 µM 5-AZA for 48 h. Hence, further work focusing on the optimal conditions for CM differentiation of equine stem cells derived from adipose tissue, as well as possibly from other origins, are needed.

Cancer Stem Cell Markers in Rhabdomyosarcoma in Children.

(1) Background: The aim of the present study was to assess the cancer stem cell (CSC) markers CD24, CD44, CD133, and ALDH1A1 in rhabdomyosarcoma (RMS) in children and to define their prognostic role in this group of patients. (2) Methods: The study material was archival tissue specimens collected from 49 patients under 18 years of age and who had been diagnosed with RMS. Immunohistochemistry (IHC) was used to evaluate the expression of the selected CSC markers in the tumor tissue. Expression was evaluated using a semiquantitative IRS scale based on the one developed by Remmele and Stenger and was correlated with the clinical and pathomorphological parameters of prognostic importance in RMS. (3) Results: Expression of the selected CSC markers CD24, CD44, CD133, and ALDH1A1 was demonstrated in 83.7%, 55.1%, 81.6%, and 100% of the RMS patients, respectively. The expression of all of the assessed CSC markers was statistically significantly higher in the study group versus the control group. No significant correlation was found between the expression of the selected CSC markers and clinical and pathological prognostic factors that were analyzed. The expression of the CSC markers did not have a significant influence on RMS survival rates. (4) Conclusions: The results of the conducted study confirm the expression of selected CSC markers in rhabdomyosarcoma tissue in children. The study did not support the prognostic relevance of the expression of any of the assessed CSC markers. However, further studies are needed to fully understand the relevance of the selected CSC markers in RMS carcinogenesis.

PFKFB3 regulates cancer stemness through the hippo pathway in small cell lung carcinoma

PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High PFKFB3 expression in cancer stem cells promotes glycolysis and survival in the tumor microenvironment. Inhibition of PFKFB3 by the glycolytic inhibitor PFK158 and by shRNA stable knockdown in small cell lung carcinoma (SCLC) cell lines inhibited glycolysis, proliferation, spheroid formation, and the expression of cancer stem cell markers CD133, Aldh1, CD44, Sox2, and ABCG2. These factors are also associated with chemotherapy resistance. We found that PFK158 treatment and PFKFB3 knockdown enhanced the ABCG2-interacting drugs doxorubicin, etoposide, and 5-fluorouracil in reducing cell viability under conditions of enriched cancer stem cells (CSC). Additionally, PFKFB3 inhibition attenuated the invasion/migration of SCLC cells by downregulating YAP/TAZ signaling while increasing pLATS1 via activation of pMST1 and NF2 and by reducing the mesenchymal protein expression. PFKFB3 knockdown and PFK158 treatment in a H1048 SCLC cancer stem cell-enriched mouse xenograft model showed significant reduction in tumor growth and weight with reduced expression of cancer stem cell markers, ABCG2, and YAP/TAZ. Our findings identify that PFKFB3 is a novel target to regulate cancer stem cells and its associated therapeutic resistance markers YAP/TAZ and ABCG2 in SCLC models.

Evodiamine inhibits stemness and metastasis by altering the SOX9-β-catenin axis in non-small-cell lung cancer.

Evodiamine (EVO), a natural dietary alkaloid extracted from the roots of the Evodia rutaecarpa, has shown anticancer activities. Here, we have investigated EVO's role in inhibiting cell proliferation and migration in A549 and NCI-H522 lung cancer cells. EVO decreased the cell viability in A549 and NCI-H522 cells in a dose- and time-dependent manner. It also induced apoptosis by downregulating the expression of antiapoptotic Bcl-2 and upregulating the expression of cleaved caspase-3 and PARP. In addition, the treatment of EVO elevated the level of reactive oxygen species (ROS) generation inside the cells to induce the cell death pathways. In contrast, the pretreatment of ROS scavenger, N-acetyl cysteine, reverses the effect of EVO and attenuates cell death. Moreover, excess ROS generation in response to EVO resulted in the depletion of mitochondrial membrane potential. Furthermore, it induced DNA damage and arrested the cell cycle at the G2/M phase in A549 and NCI-H522 cells. Our study also investigated that EVO significantly suppressed tumorigenicity by inhibiting colony formation and tumorsphere formation. However, the treatment of EVO downregulated the cancer stem cell markers CD44 and CD133 in non-small-cell lung cancer. The inhibitory effect of EVO on cell invasion was mediated by altering the expression of E-cadherin, ZO-1, N-cadherin, and Vimentin. Additionally, we have revealed that EVO treatment showed downregulation of SOX9, an upstream component of β-catenin. Lastly, we have demonstrated that EVO targets the SOX9-β-catenin axis by reducing SOX9 and β-catenin expression. These findings suggested that EVO could be a promising agent for treating human lung cancer.