Triple-negative expression (ALDH1A1-/CD133-/mutant p53-) cases in lung adenocarcinoma had a good prognosis.

Cancer stem cells (CSCs) are major contributors to the malignant transformation of cells because of their capacity for self-renewal. Aldehyde dehydrogenase1A1 (ALDH1A1) and CD133 are promising candidate of CSC markers in non-small cell lung cancer (NSCLC). Furthermore, TP53 is frequently mutated in lung cancer, and the loss of its function is associated with malignant characteristics. However, the relationship between CSCs and mutant p53 in lung adenocarcinoma is not well-established. We examined the expression of ALDH1A1, CD133, and mutant p53 in lung adenocarcinoma patients and conducted a clinicopathological study. Triple-negative cases without ALDH1A1, CD133, and mutant p53 expression in lung adenocarcinoma were shown to have a much better prognosis than others. Our present results suggest that detection of CSC markers and mutant p53 by immunohistochemical staining may be effective in therapeutic strategies for lung adenocarcinoma.

Regulation of gliomagenesis and stemness through acid sensor ASIC1a.

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

Prognostic and predictive value of ALDH1, SOX2 and SSEA-4 in bladder cancer.

Transurethral resection of bladder tumor (TUR-BT) and radical cystectomy (RC) are standard treatment options for bladder cancer (BC). Neoadjuvant chemotherapy (NAC) prior to RC improves outcome of some patients but currently there are no valid biomarkers to identify patients who benefit from NAC. Presence of cancer stem cells (CSC) has been associated with poor outcome and resistance to chemotherapy in various cancers. Here we studied the expression of stem cell markers ALDH1, SOX2 and SSEA-4 with immunohistochemistry in tissue microarray material consisting of 195 BC patients treated with RC and 74 patients treated with TUR-BT followed by NAC and RC. Post-operative follow-up data of up to 22 years was used. Negative to weak cytoplasmic SOX2 staining was associated with lymphovascular invasion and non-organ confined disease. It was also associated with shortened cancer-specific survival, but the finding was not statistically significant. Contrary to previous reports, none of the other tested biomarkers were associated with cancer-specific mortality or clinicopathological characteristics. Neither were they associated with response to NAC. Despite the promising results of previously published studies, our results suggest that CSC markers ALDH1, SOX2 and SSEA-4 have little if any prognostic or predictive value in BC treated with RC.

Expression of PD-L1, cancer stem cell and epithelial-mesenchymal transition phenotype in non-small cell lung cancer.

The purpose of this study was to investigate the expression of PD-L1, cancer stem cells (CSC) markers (CD44, NANOG, and ALDH1) and epithelial-mesenchymal transition (EMT) markers and to evaluate their correlation and prognostic significance in non-small cell lung cancer (NSCLC) patients. PD-L1 protein expression was evaluated in resected 277 NSCLC cases and its correlation with CSC and EMT marker expression and survival was determined based on immunohistochemical (IHC) analysis. mRNA expression obtained from The Cancer Genome Atlas and the Kaplan-Meier plotter database were compared with the IHC results. PD-L1 expression was negatively correlated with ALDH1 expression in adenocarcinoma by IHC and mRNA expression. PD-L1 expression was also associated with mesenchymal phenotype (vimentin and TWIST) adenocarcinoma by IHC and mRNA expression. PD-L1 expression was associated with poor prognosis in adenocarcinoma by IHC. However, NANOG or ALDH1 expression measured by IHC and mRNA expression was correlated with a favourable prognosis in adenocarcinoma. Epithelial marker p120-catenin detected by IHC and mRNA expression was associated with a favourable prognosis in adenocarcinoma, however mesenchymal marker TWIST was associated with a worse prognosis. Patients with low PD-L1 and high ALDH1 expression showed more favourable prognoses than adenocarcinoma patients with other expression patterns. In multivariate analysis, ALDH1 detected by IHC and mRNA expression was an independent favourable prognostic marker for adenocarcinoma. Our study results support the hypothesis that PD-L1 interacts with CSC and EMT features and that PD-L1, ALDH1, and the mesenchymal phenotype may serve as tandem markers as prognostic factor in NSCLC.

Role of cancer stem cell markers ALDH1, BCL11B, BMI-1, and CD44 in the prognosis of advanced HNSCC.

PURPOSE: Cancer stem cells (CSCs) are held accountable for the progress of head and neck squamous cell carcinoma (HNSCC). In the presented study, the authors evaluated the prognostic value of CSC markers in two particular HNSCC cohorts. METHODS: This two cohort study consisted of 85 patients with advanced stage HNSCC, treated with primary radio(chemo)therapy (pRCT), and 95 patients with HNSCC, treated with surgery and partially adjuvant radio(chemo)therapy. Overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) were assessed. Samples were assessed for the expression of different molecular stem cell markers (ALDH1, BCL11B, BMI­1, and CD44). RESULTS: In the pRCT cohort, none of the baseline patient and tumor features exhibited a statistically significant relation with survival in either the cohort or the human papillomavirus (HPV)-stratified subcohorts. High expression of BMI­1 significantly decreased OS and DFS, while high expression of CD44 decreased all modes of survival. Multivariate analysis showed significant prognostic influence for all tested CSC markers, with high BMI­1 and CD44 decreasing survival (BMI-1: OS, DFS, DSS; CD44: OS, DFS) and high ALDH1 and BCL11B showing a beneficial effect on survival (ALDH1: OS, DFS; BCL11B: OS, DSS). In the surgical cohort, classical prognosticators such as HPV status, R1 resection, and nodal status in HPV-negative HNSCC played a significant role, but the tested CSC markers showed no significant effect on prognosis. CONCLUSION: Although validation in independent cohorts is still needed, testing for CSC markers in patients with advanced or late stage HNSCC might be beneficial, especially if many comorbidities exist or disease is irresectable. The findings might guide the development and earlier use of targeted therapies in the future.

Expression of cancer stem cell markers in breast phyllodes tumor.

BACKGROUND: Phyllodes tumor (PT) is a rare tumor showing various malignant potential. The histological grade of PT is related to clinical outcome, but its relationship between gaining of malignant potential and underlying mechanism including cancer stem cell factor was not understood yet. OBJECTIVE: The main purpose of this study was to determine the expression pattern of cancer stem cell marker in PT and to understand its clinical and pathological implications. METHODS: CD44, CD166, ALDH1, and Ki-67 immunohistochemistry were performed on a tissue microarray from 185 cases of PT specimens (138 benign, 32 borderline, 15 malignant). The immunohistochemistry result and clinicopathological parameter of each cases were compared to analyze the implications of cancer stem cell markers on PT. RESULTS: Borderline/malignant PT showed higher CD44 expression of the stromal component than benign PT (p< 0.001). In lower histologic grade PT, CD166 showed increased expression in the epithelial component (p= 0.019), but decreased in the stromal component (p< 0.001). Stromal overgrowth was rarely observed as the number of positive cancer stem cell markers increased in the epithelial component (p< 0.001). In the stromal component, the number of positive cancer stem cell markers was related to higher histologic grade (p< 0.001), and increased stromal cellularity (p< 0.001), stromal atypia (p= 0.003), and stromal mitosis (p= 0.002). In benign PT, CD44 negativity (p= 0.013) and a decreased number of positive cancer stem cell markers (p= 0.012) in the epithelial component were related to poor prognosis. CONCLUSIONS: The cancer stem cell markers, CD44 and CD166, are expressed in both the epithelial and stromal components of phyllodes tumor. Besides, ALDH1 is only expressed in stromal component. In the stromal component, expression of cancer stem cell markers increases with higher PT histologic grade. In the epithelial component, the absence of cancer stem cell marker expression is related to poor clinical prognosis.

ß1,4-Galactosyltransferase V Modulates Breast Cancer Stem Cells through Wnt/ß-catenin Signaling Pathway.

PURPOSE: Breast cancer stem cells (BCSCs) contribute to the initiation, development, and recurrence of breast carcinomas. ß1,4-Galactosyltransferase V (B4GalT5), which catalyzes the addition of galactose to GlcNAcß1-4Man of N-glycans, is involved in embryogenesis. However, its role in the modulation of BCSCs remains unknown. MATERIALS AND METHODS: The relationship between B4GalT5 and breast cancer stemness was investigated by online clinical databases and immunohistochemistry analysis. Mammosphere formation, fluorescence-activated cell sorting (FACS), and in-vivo assays were used to evaluate B4GalT5 expression in BCSCs and its effect on BCSCs. B4GalT5 regulation of Wnt/ß-catenin signaling was examined by immunofluorescence and Ricinus communis agglutinin I pull-down assays. Cell surface biotinylation and FACS assays were performed to assess the association of cell surface B4GalT5 and BCSCs. RESULTS: B4GalT5, but not other B4GalTs, was highly correlated with BCSC markers and poor prognosis. B4GalT5 significantly increased the stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and promoted the production of CD44+CD24-/low cells and the formation of mammospheres. Furthermore, B4GalT5 overexpression resulted in dramatic tumor growth in vivo. Mechanistically, B4GalT5 modified and protected Frizzled-1 from degradation via the lysosomal pathway, promoting Wnt/ß-catenin signaling which was hyperactivated in BCSCs. B4GalT5, located on the surface of a small subset of breast carcinoma cells, was not responsible for the stemness of BCSCs. CONCLUSION: B4GalT5 modulates the stemness of breast cancer through glycosylation modification to stabilize Frizzled-1 and activate Wnt/ß-catenin signaling independent of its cell surface location. Our studies highlight a previously unknown role of B4GalT5 in regulating the stemness of breast cancer and provide a potential drug target for anticancer drug development.

Rational Design of a Red Fluorescent Sensor for ALDH1A1 Displaying Enhanced Cellular Uptake and Reactivity.

High aldehyde dehydrogenase 1A1 (ALDH1A1) activity has emerged as a reliable marker for the identification of both normal and cancer stem cells. To facilitate the detection, molecular imaging, and sorting of stem cells, a green fluorescent probe based on the xanthene dye scaffold was recently developed. However, green dyes are less amenable to multicolor imaging because most commercial reagents are also green. Overcoming this limitation will enable the simultaneous tracking of multiple stem cell markers. Herein, we report the development of a red congener, red-AlDeSense. Through chemical tuning we were able to achieve excellent isoform selectivity and chemostability, a good turn on response, and enhanced cellular uptake and reactivity. Importantly, red-AlDeSense represents one of only a few turn-on sensors in the red region that use the d-PeT quenching mechanism. By employing red-AlDeSense and a green anti-CD44 antibody, we were able to demonstrate staining of these two stem cell markers is independent of one another in A549 lung adenocarcinoma cells.

Itraconazole Alters the Stem Cell Characteristics of A549 and NCI-H460 Human Lung Cancer Cells by Suppressing Wnt Signaling.

BACKGROUND Cancer stem cells (CSCs) behave as their malignant counterparts, but persist after treatment, and possess properties that allow them to interact with their environment. Itraconazole, an antifungal agent, also has a role in suppressing tumor progression, but its effects in regulating tumor cell stemness remain unclear. This study aimed to evaluate the effects of itraconazole on A549 and NCI-H460 human lung cancer cell stemness in vitro. MATERIAL AND METHODS A549 and NCI-H460 human lung cancer cells and BEAS-2B normal bronchial epithelial cells were cultured with and without itraconazole. Cell viability was evaluated. The expression of stem cell markers, CD133, ATP binding cassette subfamily G member 2 (ABCG2), and aldehyde dehydrogenase 1 (ALDH1), were measured by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Sphere-forming cells were evaluated in vitro. RESULTS Itraconazole reduced the expression of stemness molecules CD133, ABCG2, and ALDH1 in A549 and NCI-H460 human lung cancer cells, and the numbers of sphere-forming cells were reduced. However, itraconazole had little effect on cell viability but enhanced the chemosensitivity of A549 and NCI-H460 cells. Itraconazole inhibited Wnt signaling. Re-activation of Wnt signaling restored itraconazole-mediated inhibition on A549 and NCI-H460 cell stemness. CONCLUSIONS Itraconazole altered the stemness characteristics of A549 and NCI-H460 human lung cancer cells by suppressing Wnt signaling but did not affect cell viability.

Hormonal Regulation of Patient-Derived Endometrial Cancer Stem-like Cells Generated by Three-Dimensional Culture.

Low-grade and early-stage endometrial cancer usually has a favorable prognosis, whereas recurrent or metastatic disease is often difficult to cure. Thus, the molecular mechanisms underlying advanced pathophysiology remain to be elucidated. From the perspective of the origin of advanced endometrial cancer, the characterization of cancer stem-like cells (CSCs) will be the first step toward the development of clinical management. We established long-term culturable patient-derived cancer cells (PDCs) from patient endometrial tumors by spheroid cell culture, which is favorable for the enrichment of CSCs. PDC-derived xenograft tumors were generated in immunodeficient NOD/Shi-scid, IL-2RγKO Jic mice. Morphologically, PDCs derived from three distinct patient samples and their xenograft tumors recapitulated the corresponding original patient tumors. Of note, CSC-related genes including ALDH1A1 were upregulated in all of these PDCs, and the therapeutic potentiality of aldehyde dehydrogenase inhibitors was demonstrated. In addition, these PDCs and their patient-derived xenograft (PDX) models exhibited distinct characteristics on the basis of their hormone responsiveness and metastatic features. Interestingly, genes associated with inflammation and tumor immunity were upregulated by 17ß-estradiol in PDC lines with high estrogen receptor expression and were also overexpressed in secondary PDCs obtained from metastatic tumor models. These results suggest that PDC and PDX models from endometrial cancer specimens would be useful to elucidate CSC traits and to develop alternative diagnostic and therapeutic options for advanced disease.

The Prognostic Value of Cancer Stem Cell Markers (Notch1, ALDH1, and CD44) in Primary Colorectal Carcinoma.

BACKGROUND: Cancer stem cells proved to have a vital role in cell migration, invasion, metastasis, and treatment resistance of colorectal cancer (CRC) that subsequently lead to poor clinical outcomes. These stem cells may be a novel therapeutic target for the management of CRC progression. Signals of the Notch-1 pathway are responsible for acquisition of stem cell characters. ALDH1 and CD44 are usually detected in stem cells in colorectal cancer. AIM: The aims of this work are to evaluate the immunohistochemical expression of cancer stem cell markers ALDH1, Notch1, and CD44 in colorectal cancer and investigate their correlation with clinicopathological characters and patient survival. METHODS: Paraffin-embedded specimens of 70 patients with primary colorectal carcinoma were analyzed for Notch 1, ALDH1, and CD44 expressions by immunohistochemistry. RESULTS: Notch1 was mainly located in the cytoplasm of CRC tissues, rarely expressed in adjacent normal tissues. A highly statistically significant relationship was found between grading, lymphovascular invasion, the degree of lymphocytic infiltration, peritumoral budding, lymph node ratio, lymph node metastasis, and Notch1 expression (p < 0.001). There was a highly statistically significant relationship found between AJCC stage and Notch1 expression (p < 0.001). CD44 was mainly located in the cell membrane of CRC tissues. A highly statistically significant relationship was found between grading (p = 0.006), lymphovascular invasion, the degree of lymphocytic infiltration, peritumoral budding, lymph node metastasis, lymph node ratio, and CD44 expression (p < 0.001). There was a highly statistically significant relationship found between AJCC stage and CD44 expression (p < 0.001). ALDH1 was detected in the cytoplasm of the CRC tissue. A highly statistically significant relationship was found between grading, lymphovascular invasion, the degree of lymphocytic infiltration, peritumoral budding, lymph node metastasis, lymph node ratio, and ALDH1 expression (p < 0.001). There was a highly statistically significant relationship found between AJCC stage and ALDH1 expression (p < 0.001). There is a highly statistically significant direct correlation between Notch1, CD44 expression, and ALDH1 expression (p < 0.001). CONCLUSIONS: There is a substantial correlation between Notch 1, ALDH1, and CD44 as cancer stem cell markers and lymph node metastasis, advanced stage and tumor recurrence in colorectal carcinoma. CONCLUSION: Expression of stem cell markers ALDH1, Notch1, and CD44 correlates with poor prognosis in a CRC and represents an independent prognostic factor. They are associated with a feature of epithelial-mesenchymal transition evidenced by their association with high tumor burden.

Immunolocalization of Cancer Stem Cells Marker ALDH1 and its Association with Tumor Budding in Oral Squamous Cell Carcinoma.

Tumor budding is a prognostic marker for oral squamous cell carcinoma (OSCC) characterized by the presence of isolated or small clusters of neoplastic cells at the tumor invasive front. Aldehyde dehydrogenase-1 (ALDH1) is associated with tumorigenesis, linked to treatment resistance and shown to identify cancer stem cells (CSC)-like cells. This study aimed to evaluate the expression of ALDH1 and its association with tumor budding in OSCC. Immunohistochemistry was employed in 163 OSCC samples to identify pancytokeratin (AE1/AE3) and ALDH1. While pancytokeratin (AE1/AE3) identified squamous tumor buds, the CSC-like cells were identified using ALDH1. A Chi square test was used to evaluate association between ALDH1 expression and tumor budding, while McNemar's test was used to identify differences in ALDH1 expression between the budding area and the area outside the budding. A positive expression of ALDH1 was observed in 47.24% of the samples and in 70% of anatomic locations affected. No association was observed between ALDH1 expression and tumor budding (p > 0.05). In tumors with high-intensity tumor budding, ALDH1 expression was higher in the budding area than in the area outside the budding (p < 0.05). The finding that tumor bud cells in OSCC show phenotypic characteristics of CSC-like cells reinforces the relevance of tumor budding in determining the biological behavior of this malignant neoplasm. Moreover, the presence of CSC-like cells in nearly half of evaluated samples of OSCC and in most of the affected anatomic locations is in accordance with the CSC model of oral carcinogenesis.

Prognostic Value of Cancer Stem Cell Markers in Potentially Malignant Disorders of Oral Mucosa: A Meta-analysis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is usually preceded by clinically visible changes on oral mucosa categorized as oral potentially malignant disorders (OPMD). The progression of OPMD to OSCC is a multistep process that provides an opportunity for early cancer detection and interception. Recent research suggests that cancer stem cells (CSC) hold the key to unlocking effective strategies to curb initiation and growth of several malignant neoplasms, including OSCC. In this meta-analysis, we evaluated the efficacy of CSC markers CD133, podoplanin, ALDH1, and others in predicting risk of malignant transformation of OPMDs. METHODS: The PubMed database was systematically reviewed for relevant articles. Quality of eligible studies was assessed as per reporting recommendations for tumor marker (REMARK) criteria. A total of 18 investigations from 12 studies evaluated clinical or prognostic significance of CSC markers in OPMDs. A reasonable number of patients (1,659) were included in this analysis. RESULTS: Positive expression of CSC markers in OPMDs is significantly associated with progression to OSCC [risk ratio (RR), 3.31; 95% confidence interval (CI), 2.72-4.02]. Variability in CSC population makes it difficult to understand exact biology of OPMDs based on single CSC marker investigation. CONCLUSIONS: Identifying CSC population is a reliable prognostic indicator in OPMDs with or without dysplasia. Multi-marker panel investigation for CSCs in OPMDs may assist in curtailing new cases of oral cancer to a great extent. IMPACT: The study illustrates that evaluating CSC marker expression in OPMDs is a key tool in identifying high-risk cases to prevent development of OSCC.