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Intrabdominal dissemination of malignant mesothelioma (MM) and pseudomyxoma peritonei (PMP) is poorly characterized with respect to the stemness window which malignant cells activate during their reshaping on the epithelial-mesenchymal (E/M) axis. To gain insights into stemness properties and their prognostic significance in these rarer forms of peritoneal metastases (PM), primary tumor cultures from 55 patients selected for cytoreductive surgery with hyperthermic intraperitoneal chemotherapy were analyzed for cancer stem cells (CSC) by aldehyde dehydrogenase 1 (ALDH1) and spheroid formation assays, and for expression of a set of plasticity-related genes to measure E/M transition (EMT) score. Intratumor heterogeneity was also analyzed. Samples from PM of colorectal cancer were included for comparison. Molecular data were confirmed using principal component and cluster analyses. Associations with survival were evaluated using Kaplan-Meier and Cox regression models. The activity of acetylsalicylic acid (ASA), a stemness modifier, was tested in five cultures. Significantly increased amounts of ALDH1 bright -cells identified high-grade PMP, and discriminated solid masses from ascitic/mucin-embedded tumor cells in both forms of PM. Epithelial/early hybrid EMT scores and an early hybrid expression pattern correlated with pluripotency factors were significantly associated with early peritoneal progression (p = .0343 and p = .0339, respectively, log-rank test) in multivariable models. ASA impaired spheroid formation and increased cisplatin sensitivity in all five cultures. These data suggest that CSC subpopulations and hybrid E/M states may guide peritoneal spread of MM and PMP. Stemness could be exploited as targetable vulnerability to increase chemosensitivity and improve patient outcomes. Additional research is needed to confirm these preliminary data., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

The goal of this paper is to discuss the role of ALDH isozymes in different cancers, review advances in ALDH1-targeting cancer therapies, and explore a mechanism that explains how ALDH expression becomes elevated during cancer development. ALDH is often overexpressed in cancer, and each isoform has a unique expression pattern and a distinct role in different cancers. The abnormal expression of ALDHs in different cancer types (breast, colorectal, lung, gastric, cervical, melanoma, prostate, and renal) is presented and correlated with patient prognosis. ALDH plays a significant role in various cellular functions, such as metabolism, oxidative stress response, detoxification, and cellular differentiation. Among the ALDH families, ALDH1 has gained considerable attention as a cancer stem cell (CSC) marker due to its significant role in the maintenance of stemness and the differentiation of stem cells (SCs), along with its involvement in tumorigenesis. A description of the cellular mechanisms and physiology of ALDH1 that underlies cancer development is provided. Moreover, current advances in ALDH1-targeting cancer therapies are discussed., Competing Interests: The authors do not have any conflicts of interest.

Gene fusions are common somatic alterations in cancers, and fusions with tumorigenic features have been identified as novel drivers of cancer and therapeutic targets. Few studies have determined whether the oncogenic ability of fusion genes is related to the induction of stemness in cells. Cancer stem cells (CSCs) are a subset of cells that contribute to cancer progression, metastasis, and recurrence, and are critical components of the aggressive features of cancer. Here, we investigated the CSC-like properties induced by CD63-BCAR4 fusion gene, previously reported as an oncogenic fusion, and its potential contribution for the enhanced metastasis as a notable characteristic of CD63-BCAR4. CD63-BCAR4 overexpression facilitates sphere formation in immortalized bronchial epithelial cells. The significantly enhanced sphere-forming activity observed in tumor-derived cells from xenografted mice of CD63-BCAR4 overexpressing cells was suppressed by silencing of BCAR4. RNA microarray analysis revealed that ALDH1A1 was upregulated in the BCAR4 fusion-overexpressing cells. Increased activity and expression of ALDH1A1 were observed in the spheres of CD63-BCAR4 overexpressing cells compared with those of the empty vector. CD133 and CD44 levels were also elevated in BCAR4 fusion-overexpressing cells. Increased NANOG, SOX2, and OCT-3/4 protein levels were observed in metastatic tumor cells derived from mice injected with CD63-BCAR4 overexpressing cells. Moreover, DEAB, an ALDH1A1 inhibitor, reduced the migration activity induced by CD63-BCAR4 as well as the sphere-forming activity. Our findings suggest that CD63-BCAR4 fusion induces CSC-like properties by upregulating ALDH1A1, which contributes to its metastatic features., (© 2024 The Author(s). Molecular Carcinogenesis published by Wiley Periodicals LLC.)

Since metastasis accounts for the majority of cancer morbidity and mortality, attempts are focused to block metastasis and metastasis initiating cellular programs. It is generally believed that hypoxia, reactive oxygen species (ROS) and the dysregulated redox pathways regulate metastasis. Although induction of epithelial to mesenchymal transition (EMT) can initiate cell motility to different sites other than the primary site, the initiation of a secondary tumor at a distant site depends on self-renewal property of cancer stem cell (CSC) property. That subset of metastatic cells possessing CSC property are referred to as metastasis initiating cells (MICs). Among the different cellular intermediates regulating metastasis in response to hypoxia by inducing EMT and self-renewal property, ALDH1A1 is a critical molecule, which can be used as a marker for MICs in a wide variety of malignancies. The cytosolic ALDHs can irreversibly convert retinal to retinoic acid (RA), which initiates RA signaling, important for self-renewal and EMT. The metastasis permissive tumor microenvironment increases the expression of ALDH1A1, primarily through HIF1α, and leads to metabolic reprograming through OXPHOS regulation. The ALDH1A1 expression and its high activity can reprogram the cancer cells with the transcriptional upregulation of several genes, involved in EMT through RA signaling to manifest hybrid EMT or Hybrid E/M phenotype, which is important for acquiring the characteristics of MICs. Thus, the review on this topic highlights the use of ALDH1A1 as a marker for MICs, and reporters for the marker can be effectively used to trace the population in mouse models, and to screen drugs that target MICs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This research aims to explore the mechanism by which microRNAs may regulate the biological behavior of tumor cells in ALDH1+ fibrosarcoma. We identified differentially expressed miRNAs in ALDH + NMFH-1 cells, screened genes related to sarcoma metastasis in the TCGA database, and finally obtained key genes regulated by miRNAs that are involved in metastasis. The function and mechanism of these key genes were then validated at the cellular level. Using the ULCAN database, a significant correlation was found between hsa-mir-206 and mortality in sarcoma patients. WGCNA analysis identified 352 genes related to tumor metastasis. Through Venn diagrams, we obtained 15 metastasis-related genes regulated by hsa-mir-206. Survival analysis showed that SYNPO2 expression is significantly correlated with survival rate and is significantly underexpressed in multiple tumors. SYNPO2 showed a negative correlation with macrophages and a positive correlation with CD8 + T cells. After inhibiting the expression of hsa-mir-206 with siRNA plasmids, the mRNA expression of SYNPO2 was significantly upregulated. The results of CCK8 assay, scratch assay, and transwell assay showed that the proliferation and migration ability of NFMH-1 cells were promoted after SYNPO2 was inhibited. ALDH1+ tumor stem cells promote the proliferation and invasion of malignant fibrous histiocytoma cells by inhibiting SYNPO2 through hsa-mir-206., Competing Interests: Declaration of Competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aldehyde dehydrogenase 1A1 (ALDH1A1) stands out as one of the most reliable intracellular biomarkers for stem cells because it is expressed in both cancer stem cells (CSCs) and normal somatic stem cells (NSCs). Although several turn-on fluorescent probes for ALDH1A1 have been developed to visualize CSCs in cancer cells, the discrimination of CSCs from NSCs is difficult. We here report an AND-type dual-responsive fluorescent probe, CHO_βgal, the near-infrared fluorescence of which can be turned on after responding to both ALDH1A1 and β-galactosidase. The AND-type dual responsiveness enables CSCs to be clearly visualized, whereas NSCs are non-emissive in microscopy. CSC-positive metastasis model lungs were successfully discriminated from normal lungs in ex vivo staining experiments using CHO_βgal, whereas the single-input ALDH1A1-responsive probe failed to achieve this discrimination owing to pronounced false-positive fluorescence output from lung NSCs. In tissue slice staining experiments, even in the presence of adjacent normal tissues, the peripheral region-specific localization of CSCs was clear. The versatility of CHO_βgal holds promise not only as a fundamental in vitro research tool for visualizing CSCs but also as a valuable asset in practical tissue staining diagnosis, significantly contributing to the assessment of cancer malignancy.

Drug-resistant and metastatic cancer cells such as a small population of cancer stem cells (CSCs) play a crucial role in metastasis and relapse. Conventional small-molecule chemotherapeutics, however, are unable to eradicate drug-resistant CSCs owing to limited interface inhibitory effects. Herein, it is reported that enhanced interfacial inhibition leading to eradication of drug-resistant CSCs can be dramatically induced by self-insertion of bioactive graphene quantum dots (GQDs) into DNA major groove (MAG) sites in cancer cells. Since transcription factors regulate gene expression at the MAG site, MAG-targeted GQDs exert greatly enhanced interfacial inhibition, downregulating the expression of a collection of cancer stem genes such as ALDH1, Notch1, and Bmi1. Moreover, the nanoscale interface inhibition mechanism reverses cancer multidrug resistance (MDR) by inhibiting MDR1 gene expression when GQDs are used at a nontoxic concentration (1/4 × half-maximal inhibitory concentration (IC 50 )) as the MDR reverser. Given their high efficacy in interfacial inhibition, CSC-mediated migration, invasion, and metastasis of cancer cells can be substantially blocked by MAG-targeted GQDs, which can also be harnessed to sensitize clinical cytotoxic agents for improved efficacy in combination chemotherapy. These findings elucidate the inhibitory effects of the enhanced nano-bio interface at the MAG site on eradicating CSCs, thus preventing cancer metastasis and recurrence., (© 2024 Wiley‐VCH GmbH.)

The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com ., (© 2024. The Author(s).)

Background/aim: Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDH high ) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy., Materials and Methods: Gastric CSCs/CICs were isolated as ALDH high cells using the human gastric cancer cell line, MKN-45. ALDH high clone cells and ALDH low clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDH high clone cells and ALDH low clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay., Results: Three ALDH high clone cells were isolated from MKN-45 cells. ALDH high clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells., Conclusion: High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDH high CSCs/CICs evade T cells due to lower expression of HLA class 1., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Lung cancer occupies a leading position in the structure of global cancer morbidity and mortality, due to the biological properties of the key pool of tumor cells - cancer stem cells (CSCs). The effects of SARS-CoV2 on tumor CSCs and its niche have not been studied., Objective: To study CSCs in lung adenocarcinomas after COVID19., Material and Methods: Surgical material from lung adenocarcinoma from 12 patients who had a new coronavirus infection and from 12 patients who did not have COVID19 was examined. Analysis of clinical and anamnestic data, macroscopic and microscopic examination of tumor samples and adjacent intact tissue, immunohistochemical reactions using antibodies to virus proteins and CSC markers ALDH1, CD133 and CD34 were performed., Results: Adenocarcinoma samples from patients in the main group showed a significant increase in the number of cells expressing the CSCs markers ALDH1, CD133 and CD34 compared to adenocarcinoma samples from control group patients without SARS-CoV2 infection. We found an increase in the number of CSCs in patients with adenocarcinoma metastasis in lymph nodes in both the main and control groups. CSCs of lung adenocarcinomas from SARS-CoV2 survivors contain virus proteins Nucleocapside and Spike protein., Conclusions: We found an increase in the number of CSCs with expression of ALDH1, CD133 and CD34 in lung adenocarcinoma in patients with new coronavirus infection. Increased number of ALDH1+, CD133+ CD34+ CSCs in tumor tissue enhance the metastatic potential of lung adenocarcinoma. The Nucleocapsid and Spike proteins of SARS-CoV2 virus are detectable in lung tissue from patients with new coronavirus infection, both in adenocarcinoma cells, CSCs, and in type II pneumocytes, macrophages, and endothelial cells, suggesting prolonged persistence of the virus proteins and probably the virus.

Background/aim: This study aimed to examine the clinical significance of the protein expression of the cancer stem cell (CSC) markers ALDH1A1, CD133, CD44, and MSI-1 in primary and metastatic tissues of patients with breast cancer (BC)., Patients and Methods: ALDH1A1, CD133, CD44, and MSI-1 protein expression in pairs of primary and metastatic tissues of 55 patients with BC with metastases treated at Kanagawa Cancer Center between January 1970 and December 2016 were evaluated using immunohistochemical assay and their association with clinicopathological factors and survival was examined., Results: There were no significant differences in CSC marker expression rates between primary and metastatic tissues for any CSC markers. Regarding the relationship between CSC marker expression in primary tissues and survival, patients with high CD133 expression had significantly lower recurrence-free survival (DFS) and overall survival. On multivariate analysis, they were also a poor independent predictor of DFS (hazard ratio=4.993, 95%CI=2.189-11.394, p=0.0001). In contrast, there was no significant association between the expression of any CSC marker in metastatic tissues and survival., Conclusion: CD133 expression in the primary BC tissue may be a useful risk factor for recurrence in patients with BC., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. Cancer stem cells (CSCs) are seeds for tumor relapse and metastasis. However, pathways that maintain stemness genes are not fully understood. Here, we report that the enzyme euchromatic histone lysine methyltransferase 1 (EHMT1) is expressed in primary and relapse ARMS tumors. EHMT1 suppression impaired motility and induced differentiation in ARMS cell lines and reduced tumor progression in a mouse xenograft model in vivo. RNA sequencing of EHMT1-depleted cells revealed downregulation of ALDH1A1 that is associated with CSCs. Consistent with this, inhibition of ALDH1A1 expression and activity mimicked EHMT1 depletion phenotypes and reduced tumorsphere formation. Mechanistically, we demonstrate that EHMT1 does not bind to the ALDH1A1 promoter but activates it by stabilizing C/EBPβ, a known regulator of ALDH1A1 expression. Our findings identify a role for EHMT1 in maintenance of stemness by regulating ALDH1A1 expression and suggest that targeting ALDH + cells is a promising strategy in ARMS. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background/aim: Aldehyde dehydrogenase (ALDH) 1A1 is a well-known marker for cancer stem cells (CSCs), characterized by self-renewal capacity and multidrug resistance in breast cancer. We developed a near-infrared turn-on fluorescence probe for ALDH1A1, C5S-A, which is suitable for observing and analyzing viable cells. Here, we demonstrated the utility of C5S-A in CSC research using breast cancer cell lines., Materials and Methods: To evaluate concordance between C5S-A and conventional stem cell markers, breast cancer cells sorted for ALDEFLUOR-positive cells and for CD44 + /CD24 - cell populations were stained with C5S-A. Tumorigenicity of C5S-A-positive cells was examined by mammosphere formation assay and subcutaneous transplantation to immunodeficient mice. Additionally, to determine how long fluorescence from a single staining remained observable, we cultured breast cancer cells for 5 days after C5S-A staining. We then evaluated whether C5S-A-positive cells possessed resistance to cytotoxic drugs by chronological imaging., Results: C5S-A staining showed good concordance with conventional breast CSC markers, and good utility for research into CSC characteristics in breast cancer cell lines, including tumorigenesis. Additionally, C5S-A was observable for more than 3 days with a single staining. Using this property, we then confirmed that C5S-A-positive cells possessed resistance to cytotoxic drugs, which is one of the characteristics of CSCs., Conclusion: We showed that C5S-A is suitable for CSC research using breast cancer cell lines, and confirmed its utility in observing cells over time., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Tumor-initiating cells (TIC) are associated with tumor initiation, growth, metastasis, and recurrence. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a TIC marker in many cancers, including breast cancer. However, the molecular mechanisms underlying ALDH1A1 functions in solid tumors remain largely unknown. Here we demonstrate that ALDH1A1 enzymatic activity facilitates breast tumor growth. Mechanistically, ALDH1A1 decreased the intracellular pH in breast cancer cells to promote phosphorylation of TAK1, activate NFκB signaling, and increase the secretion of GM-CSF, which led to myeloid-derived suppressor cell expansion and immunosuppression. Furthermore, the ALDH1A1 inhibitor disulfiram and chemotherapeutic agent gemcitabine cooperatively inhibited breast tumor growth and tumorigenesis by purging ALDH + TICs and activating T-cell immunity. These findings elucidate how active ALDH1A1 modulates the immune system to promote tumor development, highlighting new therapeutic strategies for malignant breast cancer. SIGNIFICANCE: ALDH1A1 enzyme activity induces MDSC expansion and triggers a procancer immune microenvironment to facilitate breast cancer progression, providing a novel therapeutic vulnerability in this disease., (©2021 American Association for Cancer Research.)

Background/aim: We examined the inhibitory effects of both glyoxalase 1 (GLO 1) and protein kinase C (PKC)λ in aldehyde dehydrogenase 1 (ALDH1)-positive breast cancer stem cells (CSCs)., Materials and Methods: Breast cancer genomics datasets (TCGA, n=593; METABRIC, n=1904) were downloaded and statistically analyzed. The effects of GLO 1 and PKCλ on trypan blue staining and tumor-sphere formation by ALDH1 high cells derived from triple negative breast cancer (TNBC) and basal-like breast cancer were examined., Results: GLO 1 high , PKCλ high , and ALDH1A3 high tumors were enriched in stage I/II/III/IV samples, associated with the HER2 and TNBC subtypes according to receptor status, and associated with the HER2-enriched and basal-like subtypes according to PAM50. Inhibition of either GLO 1 (TLSC702) or PKCλ (ANF) suppressed tumor-sphere formation and enhanced death in ALDH1 high cells. TLSC702 also effectively inhibited tumor-sphere formation and induced death in PKCλ knockout ALDH1 high cells., Conclusion: GLO 1 and PKCλ are important for the survival of ALDH1-positive breast CSCs, and may represent potential therapeutic targets for the treatment of ALDH1-positive breast CSCs., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objectives: Prognostic biomarkers in cervical cancer are widely investigated, including cancer stem cell (CSC) markers. However, their significance remains uncertain. This study aimed to determine the role of cervical cancer stem cell (CCSC) markers for survival., Materials and Methods: We conducted a systematic review and meta-analysis (PROSPERO CRD42021237072) of studies reporting CCSC markers as the prognostic predictor based on PRISMA guidelines. We included English articles investigating associations of CCSCs expression in tissue tumor with overall survival (OS) or disease-free survival (DFS) from PubMed, EBSCO, and The Cochrane Library databases. The quality of studies was analyzed based on Newcastle-Ottawa Quality Assessment Scale., Results: From 413 publications, after study selection with inclusion and exclusion criteria, 22 studies were included. High expressions of CCSC markers were associated with poor OS and DFS (HR= 1.05, 95% CI: 1.03 - 1.07, P <0.0001; HR= 1.31, 95% CI: 1.09 - 1.17, P <0.00001; respectively). Sub-analysis of individual CCSC markers indicated significant correlations between CD44 (HR= 1.14, 95% CI: 1.07 - 1.22, P 0.0001), SOX2 (HR= 1.58, 95% CI: 1.17 - 2.14, P 0.003), OCT4 (HR= 1.03, 95% CI: 1.01 - 1.06, P 0.008), ALDH1 (HR= 1.36, 95% CI: 1.13 - 1.64, P 0.001), and CD49f (HR= 3.02, 95% CI: 1.37 - 6.64, P 0.006) with worse OS; OCT4 (HR= 1.14, 95% CI 1.06 - 1.22, P 0.0003), SOX2 (HR= 1.11, 95% CI: 1.06 - 1.16, P <0.0001), and ALDH1 (HR= 1.22, 95% CI: 1.10 - 1.35, P 0.0002) with poor DFS. We did not conduct a meta-analysis for MSI-1 and CK17 because only one study investigated those markers., Conclusion: Expressions of OCT4, SOX2, and ALDH1 were associated with poor OS and DFS in cervical cancer tissue. These markers might have potential roles as prognostic biomarkers to predict unfavorable survival.

Activating molecule in Beclin-1-regulated autophagy (AMBRA1), a negative regulator of tumorigenesis, is a substrate receptor of the ubiquitin conjugation system. ALDH1B1, an aldehyde dehydrogenase, is a cancer stem cell (CSC) marker that is required for carcinogenesis via upregulation of the β-catenin pathway. Although accumulating evidence suggests a role for ubiquitination in the regulation of CSC markers, the ubiquitination-mediated regulation of ALDH1B1 has not been unraveled. While proteome analysis has suggested that AMBRA1 and ALDH1B1 can interact, their interaction has not been validated. Here, we show that AMBRA1 is a negative regulator of ALDH1B1. The expression of ALDH1B1-regulated genes, including PTEN , CTNNB1 (β-catenin), and CSC-related β-catenin target genes, is inversely regulated by AMBRA1, suggesting a negative regulatory role of AMBRA1 in the expression of ALDH1B1-regulated genes. We found that the K27- and K33-linked ubiquitination of ALDH1B1 is mediated via the cooperation of AMBRA1 with other E3 ligases, such as TRAF6. Importantly, ubiquitination site mapping revealed that K506, K511, and K515 are important for the K27-linked ubiquitination of ALDH1B1, while K33-linked ubiquitination occurs at K506. A ubiquitination-defective mutant of ALDH1B1 increased the self-association ability of ALDH1B1, suggesting a negative correlation between the ubiquitination and self-association of ALDH1B1. Together, our findings indicate that ALDH1B1 is negatively regulated by AMBRA1-mediated noncanonical ubiquitination.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification., (©2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Aldehyde dehydrogenase 1 member A1 (ALDH1A1) is one of the most well studied breast cancer stem cells. Its expression has been associated with poor clinicopathological features and clinical outcomes in several studies. This paper studies the expression of ALDH1A1 and its combination with CD44 + /CD24 -/low breast cancer stem cell and their association with clinicopathological parameters and molecular subtypes., Method: Tissue Microarray was constructed from 222 Formalin Fixed Paraffin Embedded (FFPE) breast cancer tissues. The expression of ALDH1A1, CD44 and CD24 were assessed by Immunohistochemistry (IHC). The association of ALDH1A1 and its association with clinicopathological parameters, molecular subtypes, CD44 and CD24 were studied in an African population. The association between CD44 + /CD24 -/low /ALDH1 + and the clinicopathological phenotypes were also studied., Results: A high ALDH1A1 expression of 90% was recorded in this study. No association was found between ALDH1A1 and clinicopathological parameters. ALDH1A1 was positively associated with CD24 (r = 0.228, OR-4.599 95% CI- 1.751-12.076, p = 0.001) and CD44 (r = 0.228, OR-5.538 95%CI- 1.841-16.662, p = 0.001) but not associated with CD44 + /CD24 -/low (r = 0.134, OR- 2.720 95%CI- 0.959-7.710, p = 0.052). CD44 + /CD24 - /ALDH1 + however had significant associations with Age (p- 0.020, r = 0.161, OR- 2.771, 95%CI 1.147-6.697), Gender (p = 0.004, OR- 15.333 95%CI 1.339-175.54), Tumour grade (p = 0.005, r = 0.197, OR-3.913 95%CI 1.421-10.776) and clinical prognostic staging (p = 0.014, r = 0.182, OR-3.028 95%CI- 1.217-7.536). There was no association between CD44 + /CD24 - /ALDH1 + and the molecular subtypes., Conclusion: The high expression of ALDH1A1 in breast cancer makes it an important target for targeted therapy. This study further confirms the increased tumourigenicity of CD44 + /CD24 - /ALDH1 + combination phenotype and its association with increased tumour grade and clinical prognostic stage. Survival studies of ALDH1A1 and other breast cancer stem cells in African populations are strongly recommended to help further understand their effect on tumour aggressiveness., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We previously demonstrated that immunohistochemistry for γ-H2AX, a biomarker of DNA damage, is useful for early detection of urinary bladder carcinogens in rats. In a 28-day repeated-dose study, γ-H2AX was shown to have high sensitivity for detection of bladder carcinogens. However, no reports have evaluated whether a combination of multiple biomarkers may further improve sensitivity. Accordingly, in this study, we aimed to evaluate the applicability of bladder tissue and cancer stem cell markers, including cytokeratin 14 (KRT14), aldehyde dehydrogenase 1A1 (ALDH1A1), and cluster of differentiation 44 (CD44), as complementary markers for early detection of bladder carcinogens. Bladder samples obtained from male F344 rats orally treated with 14 bladder carcinogens and five nonbladder carcinogens for 28 days were used for immunohistochemical analysis of stem cell markers. In the bladder carcinogen-treated rats, increases in KRT14, ALDH1A1, and CD44 expression were observed in 9, 10, and 10 out of 14 groups, respectively, whereas the five nonbladder carcinogens did not cause upregulation of these markers. Although most epithelial cells with KRT14 or ALDH1A1 expression were also positive for CD44, KRT14 and ALDH1A1 expression were mutually exclusive. Twelve bladder carcinogens showed increases in at least one of the three markers, indicating that the combined evaluation showed higher sensitivity than the use of individual markers alone. Importantly, two of three bladder carcinogens that did not induce γ-H2AX immunostaining showed stem cell marker expression. Our results demonstrated that these stem cell markers may be useful as complementary markers for γ-H2AX in evaluation of bladder carcinogens.

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Cancer stem cells (CSC) are cells that exhibit stem cell properties and are pivotal in tumor biology. CSC markers have been described for many tumorous entities. However, to this date, there is no data on CSC markers in respect to squamous cell carcinomas (SCC) of the salivary glands., Methods: Histologic samples from patients with salivary gland SCCs were stained for CSC markers (ALDH-1/BMI-1/SOX-2/CD-44/vimentin) and divided into high and low expression subgroups. These were then correlated with tumor and patient characteristics as well as overall survival (OS), disease-specific survival, recurrence-free survival and local control rates (LCR) after 3 and 5 years., Results: Overall, 31 samples were included. CD-44 and ALDH-1 expression were associated with tumor origin (metastatic/primary disease, p = 0.048 and p = 0.011, respectively). Strong BMI-1 expression was associated with poorer OS (62.9 vs. 27.3%, p = 0.029), strong SOX-2 expression was associated with poorer LCR (62.5 vs. 21.9%, p = 0.007)., Conclusion: CD-44 and ALDH-1 may be useful in differentiating between primary SCCs and metastatic disease. BMI-1 and SOX-2 are correlated with poorer prognosis., (© 2021 S. Karger AG, Basel.)

Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from Kras G12D ; Pdx1-Cre (KC) and Kras G12D ; Trp53 R172H ; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid β-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid β-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.

The aim of this study was to investigate the role of myeloid-derived suppressor cells (MDSC) in the induction of cancer stem-like cells (CSC) and programmed death ligand 1 (PD-L1) expression in ovarian cancer. CSC were defined as tumor cells expressing high levels of aldehyde dehydrogenase 1 (ALDH 1). We inoculated G-CSF-expressing or Mock-expressing ovarian cancer cells into mice, and the frequencies of MDSC and CSC in tumors of these models were compared by flow cytometry. To directly demonstrate the role of MDSC in the induction of CSC and the increase in PD-L1 expression, we performed in vitro co-culture. MDSC and CSC (ALDH-high cells) were more frequently observed in G-CSF-expressing cell-derived tumors than in Mock-expressing cell-derived tumors. Co-culture experiments revealed that MDSC increased the number of CSC via the production of PGE2. Moreover, PGE2 produced by MDSC increased tumor PD-L1 expression via the mammalian target of rapamycin (mTOR) pathway in ovarian cancer cells. In an in vitro experiment in which ovarian cancer cells were co-cultured with MDSC, higher expression of PD-L1 was observed in CSC than in non-CSC (ALDH-low cells). Furthermore, by immunofluorescence staining, we found that PD-L1 was co-expressed with ALDH1 in in vivo mouse models. In conclusion, PGE2 produced by MDSC increases the stem cell-like properties and tumor PD-L1 expression in epithelial ovarian cancer. Depleting MDSC may be therapeutically effective against ovarian cancer by reducing the number of CSC and tumor PD-L1 expression.

Digestive system cancers are the most frequent cancers worldwide and often associated with poor prognosis because of their invasive and metastatic characteristics. Recent studies have found that the plasticity of cancer cells can impart cancer stem-like properties via the epithelial-mesenchymal transition (EMT). Cancer stem-like properties such as tumor initiation are integral to the formation of metastasis, which is the main cause of poor prognosis. Numerous markers of cancer stem cells (CSCs) have been identified in many types of cancer. Therefore, CSCs, via their stem cell-like functions, may play an important role in prognosis after surgery. While several reports have described prognostic analysis using CSC markers, few reviews have summarized CSCs and their association with prognosis. Herein, we review the prognostic potential of eight CSC markers, CD133, CD44, CD90, ALDH1A1, EPCAM, SOX2, SOX9, and LGR5, in digestive cancers including those of the pancreas, colon, liver, gastric, and esophagus.

A study was to investigate the regulation of bone marrow mesenchymal stem cells (BMSCs) on the stemness of hypopharyngeal cancer cells (FaDu cells). Green fluorescent protein-labeled FaDu cells were cocultured with BMSCs and then were isolated. In vitro experiments, including cell cycle and apoptosis analyses and clonogenic and sphere formation assays, were conducted using the cocultured FaDu cells to determine the stemness of FaDu cells. The tumor formation assay was performed through subcutaneous injection of FaDu cells into nude mice to determine the tumorigenic ability of FaDu cells after coculture. Immunohistochemical analysis of CD44 and ALDH1 was performed on the tumor tissue. After coculturing with human BMSCs, the ratio of FaDu cells at G2 phase was increased, while the ratios at S and G1 phases were decreased. In addition, coculture reduced apoptosis, but increased the clonogenic ability and sphere formation efficiency of FaDu cells. Finally, coculturing FaDu cells induced more robust and faster tumor formation as well as increased expression levels of CD44 and ALDH1 in tumor tissue. BMSCs promote the stemness of hypopharyngeal cancer cells.

Purpose: Breast cancer stem cells (BCSCs) contribute to the initiation, development, and recurrence of breast carcinomas. β1,4-Galactosyltransferase V (B4GalT5), which catalyzes the addition of galactose to GlcNAcβ1-4Man 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/β-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/β-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/β-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.

Background: Putative castration-resistant (CR) stem-like cells (CRSC) have been identified based on their ability to initiate and drive prostate cancer (PCa) recurrence following castration in vivo. Yet the relevance of these CRSC in the course of the human disease and particularly for the transition from hormone-naive (HN) to castration-resistance is unclear. In this study, we aimed at deciphering the significance of CRSC markers in PCa progression., Methods: We constructed a tissue microarray comprising 112 matched HN and CR tissue specimens derived from 55 PCa patients. Expression of eight stemness-associated markers (ALDH1A1, ALDH1A3, ALDH3A1, BMI1, NANOG, NKX3.1, OCT4, SOX2) was assessed by immunohistochemistry and scored as a percentage of positive tumor cells. For each marker, the resulting scores were statistically analyzed and compared to pathological and clinical data associated with the samples. Unsupervised clustering analysis was performed to stratify patients according to the expression of the eight CRSC markers. Publicly-available transcriptional datasets comprising HN and CR PCa samples were interrogated to assess the expression of the factors in silico., Results: Immunohistochemical assessment of paired samples revealed atypical patterns of expression and intra- and intertumor heterogeneity for a subset of CRSC markers. While the expression of particular CRSC markers was dynamic over time in some patients, none of the markers showed significant changes in expression upon the development of castration resistance (CR vs HN). Using unsupervised clustering approaches, we identified phenotypic subtypes based on the expression of specific stem-associated markers. In particular, we found (a) patterns of mutual exclusivity for ALDH1A1 and ALDH1A3 expression, which was also observed at the transcriptomic level in publicly-available PCa datasets, and (b) a phenotypic cluster associated with more aggressive features. Finally, by comparing HN and CR matched samples, we identified phenotypic cluster switches (ie, change of phenotypic cluster between the HN and CR state), that may be associated with clinical and predictive relevance., Conclusions: Our findings indicate stemness-associated patterns that are associated with the development of castration-resistance. These results pave the way toward a deeper understanding of the relevance of CRSC markers in PCa progression and resistance to androgen-deprivation therapy., (© 2020 Wiley Periodicals LLC.)

Estrogen-receptor-positive breast tumors are treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer stem cells (CSCs) with high aldehyde dehydrogenase activity (ALDH + cells) are enriched following AE treatment. Here, we show that the interleukin-1β (IL-1β) signaling pathway is activated in ALDH + cells, and data from single cells reveals that AE treatment selects for IL-1 receptor (IL1R1)-expressing ALDH + cells. Importantly, CSC activity is reduced by an IL1R1 inhibitor in AE-resistant models. Moreover, IL1R1 expression is increased in the tumors of patients treated with AE therapy and predicts treatment failure. Single-cell gene expression analysis revealed that at least two subpopulations exist within the ALDH + population, one proliferative and one quiescent. Following AE therapy the quiescent population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Targeting of ALDH + IL1R1 + cells merits testing as a strategy to combat AE resistance in patients with residual disease., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction., Competing Interests: The authors have declared that no competing of interests exist.

Genomic analyses have recently discovered the malignant subtype of human intrahepatic cholangiocarcinoma (ICC) characterized by frequent mutations of chromatin remodeling gene ARID1A; however, the biological and molecular functions still remain obscure. We here examined the clinical and biological significances of ARID1A deficiency in human ICC. Immunohistochemical analysis demonstrated that the loss of ARID1A was an independent prognostic factor for overall survival of ICC patients (P = 0.023). We established ARID1A-knockout (KO) cells by using the CRISPR/Cas9 system from two human cholangiocarcinoma cell lines. ARID1A-KO cells exhibited significantly enhanced migration, invasion, and sphere formation activity. Microarray analysis revealed that ALDH1A1, a stemness gene, was the most significantly elevated genes in ARID1A-KO cells. In addition, ALDH enzymatic activity as a hallmark of cancer stem cells was markedly high in the KO cells. ARID1A and histone deacetylase 1 were directly recruited to the ALDH1A1 promoter region in cholangiocarcinoma cells with undetectable ALDH1A1 expression by chromatin immunoprecipitation assay. The histone H3K27 acetylation level at the ALDH1A1 promoter region was increased in cells when ARID1A was disrupted (P < 0.01). Clinically, inverse correlation between ARID1A and ALDH1A1 expression was also identified in primary ICC (P = 0.018), and ARID1A-negative and ALDH1A1-positve ICCs showed worse prognosis than only ARID1A-negative cases (P = 0.002). In conclusion, ARID1A may function as a tumor suppressor in ICC through transcriptional downregulation of ALDH1A1 expression with decreasing histone H3K27 acetylation. Our studies provide the basis for the development of new epigenetic approaches to ARID1A-negative ICC. Immunohistochemical loss of ARID1A is an independent prognostic factor in intrahepatic cholangiocarcinoma patients. ARID1A recruits HDAC1 to the promoter region of ALDH1A1, a stemness gene, and epigenetically suppresses ALDH1A1 expression with decreasing histone H3K27 acetylation in cholangiocarcinoma cells., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Breast cancer is the most common cancer with an increasing incidence in Asia. About 20% of all breast cancers are triple-negative breast cancers (TNBCs). BCSC is a subset of tumor cells that has stem cell-like characteristics, such as a high capacity for self-renewal and tumor initiation, which implies that BCSC may cause aggressiveness of TNBC. ALDH1 has a role in early stem cell differentiation through its function in the oxidation of retinol to retinoic acid, proposed to be a strong candidate for breast cancer stem cells. Various studies have shown that ALDH1 is one of the markers of CSC that can be used as a prognosis indicator because it can be a biological marker for poor prognostic factors in TNBC. This study assessed the prognostic survival rate with a retrospective cohort method in TNBC patients. A total of 54 of 55 patients treated at RSCM were tested for the expression of ALDH1 through an immunohistochemistry assay of breast cancer tissue using ALDH1 staining. Survival analysis was done to obtain the prognostic data of ALDH1. Positive ALDH1 expression was obtained at 38.89% in TNBC patients. One-year survival and three years of survival in TNBC patients with positive ALDH1 expression were 42.9% and 33.3%, respectively. In this study, ALDH1 can be used as a poor survival prognostic factor with HR 2.636 and p value 0.013. The conclusion of this study is that ALDH1 can be used as a poor prognostic factor in TNBC patients although it cannot be an independent prognostic factor., Competing Interests: The researchers declare that there are no conflicts of interest in this study., (Copyright © 2020 Sonar Soni Panigoro et al.)

Chondrosarcoma is the second most common primary malignant bone tumor and is resistant to chemotherapy and radiation. Inadequate treatment response and poor prognosis requires novel therapeutic approaches. Proline‑rich polypeptide‑1 (PRP‑1), synthesized by brain neurosecretory cells, has demonstrated antitumor properties in JJ012‑cells; however, its underlying molecular mechanism remains unclear. The present study aimed to investigate the epigenetic regulation by which PRP‑1 inhibits chondrosarcoma cancer stem cell (CSC) proliferation and to elucidate additional CSC biomarkers in human chondrosarcoma other than ALDH1A1. Human chondrosarcoma JJ012‑cells were treated with PRP‑1 prior to performing an Aldefluor™ assay and fluorescence‑activated cell sorting in order to determine aldehyde dehydrogenase (ALDH) expression levels and isolate ALDHhigh and ALDHlow cell populations. ALDH is an established marker of CSCs in several neoplasms, including chondrosarcoma. The cells were collected and lysed for gel electrophoresis, followed by western blot analysis. The Aldefluor™ assay was used to assess the expression levels of well‑established CSC biomarkers, including CD133, CD4, CD10, CD144, CD177, CD221, CD271, leucine‑rich repeat‑containing G protein‑coupled receptor 5, SOX2 and B lymphoma Mo‑MLV insertion region 1 homolog (BMI‑1), within the ALDHhigh population of JJ012 cells. The results confirmed that ALDHA1 was the biomarker for chondrosarcoma CSCs. PRP‑1 was demonstrated to inhibit the ALDHhigh population colony and sarcosphere formation; 5 µg/ml PRP‑1 was indicated to be the optimum concentration in eliminating colonies formed by JJ012 cells (92%, P<0.001) and by the ALDHhigh CSC‑population (80.5%, P<0.001) in the clonogenic dose‑response assay. Spheroid growth unequivocally decreased with an increase in PRP‑1 dose. In order to determine the molecular mechanism by which PRP‑1 decreased the CSC population, the regulation of the mammalian Switch/sucrose non‑fermenting (SWI/SNF) complex, also referred to as BRG1‑associated factor (BAF) complex, which either activates or represses transcription, thus acting as an oncogene or tumor suppressor in human cells, was analyzed. PRP‑1 was demonstrated to decrease the expression levels of BRG, BAF170 and BRM; therefore, in JJ012 cells, these key players of the SWI/SNF (BAF) complex served an oncogenic role. The results of the present study demonstrated that PRP‑1 targets chromatin‑remodeling complexes; therefore, future efforts will be directed towards determining the interconnection between CSC maintenance, self‑renewal capacity and BAF complexes.

One of the reasons for recurrence following treatment of high grade serous ovarian carcinoma (HGSOC) is the persistence of residual cancer stem cells (CSCs). There has been variability between laboratories in the identification of CSC markers for HGSOC. We have identified new surface markers (CD24, CD9 and EPHA1) in addition to those previously known (CD44, CD117 and CD133) using a bioinformatics approach. The expression of these surface markers was evaluated in ovarian cancer cell lines, primary malignant cells (PMCs), normal ovary and HGSOC. There was no preferential expression of any of the markers or a combination. All the markers were expressed at variable levels in ovarian cancer cell lines and PMCs. Only CD117 and CD9 were expressed in the normal ovarian surface epithelium and fallopian tube. Both ALDEFLUOR (ALDH1A1) and side population assays identified a small proportion of cells (<3%) separately that did not overlap with little variability in cell lines and PMCs. All surface markers were co-expressed in ALDH1A1+ cells without preference for one combination. The cell cycle analysis of ALDH1A1+ cells alone revealed that majority of them reside in G0/G1 phase of cell cycle. Further separation of G0 and G1 phases showed that ALDH1A1+ cells reside in G1 phase of the cell cycle. Xenograft assays showed that the combinations of ALDH1A1 + cells co-expressing CD9, CD24 or EPHA1 were more tumorigenic and aggressive with respect to ALDH1A1-cells. These data suggest that a combined approach could be more useful in identifying CSCs in HGSOC., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an oncofetal protein expressed in various cancers including leukemia. In this study, we assessed the role of IGF2BP1 in orchestrating leukemia stem cell properties. Tumor-initiating potential, sensitivity to chemotherapeutic agents, and expression of cancer stem cell markers were assessed in a panel of myeloid, B-, and T-cell leukemia cell lines using gain- and loss-of-function systems, cross-linking immunoprecipitation (CLIP), and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) techniques. Here, we report that genetic or chemical inhibition of IGF2BP1 decreases leukemia cells' tumorigenicity, promotes myeloid differentiation, increases leukemia cell death, and sensitizes leukemia cells to chemotherapeutic drugs. IGF2BP1 affects proliferation and tumorigenic potential of leukemia cells through critical regulators of self-renewal HOXB4 and MYB and through regulation of expression of the aldehyde dehydrogenase, ALDH1A1. Our data indicate that IGF2BP1 maintains leukemia stem cell properties by regulating multiple pathways of stemness through transcriptional and metabolic factors.

Background: The enrichment of cancer stem cell-like cells (CSCs) has been considered to be responsible for tumor progression after an initial response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) in patients with non-small cell lung adenocarcinoma (NSCLC/ADC). CSCs with ALDH1A1 bright /CD44 high expression contribute to the TKIs resistance in NSCLC/ADC cells. All-trans retinoic acid (ATRA) has been shown to be a potential targeted therapy against CSCs due to its ability to inhibit ALDH1A1 activity. We therefore investigated whether ATRA could circumvent the resistance to improve the response to gefitinib in NSCLC/ADC cells., Methods: Treatment of NSCLC/ADC A549 and H1650 cells with gefitinib enriched the gefitinib surviving cells (GSCs). The expression of ALDH1A1 and CD44 and the IC50 values for gefitinib were determined by flow cytometry (FCM) and crystal violet assay in GSCs and ATRA-treated GSCs, respectively. Using DEAB as the positive control, direct inhibitory effect of ATRA on ALDH1A1 activity was determined by ALDEFLUOR assay, RESULTS: GSCs showed higher expression of ALDH1A1 and CD44 and IC50 values for gefitinib than their respective parental cells, suggesting that gefitinib can lead to propagation of CSC-enriched gefitinib-resistant cells. Treatment with ATRA was found to significantly reduce the increased expression of ALDH1A1 and CD44 and the IC50 values for gefitinib in A549GSC and H1650GSC cells, and ATRA could directly inhibit active ALDH1A1 as compared to DEAB., Conclusion: Our findings suggest that combination treatment with ATRA prevents gefitinib-induced enrichment of ALDH1A1 bright /CD44 high CSCs and enhances gefitinib-induced growth inhibition of NSCLC/ADC cells.

Aim : CC chemokine receptor 9 (CCR9) interacts with its exclusive ligand CCL25, resulting in promoting tumor progression and metastasis. However, the effect and mechanisms of CCR9 on lung adenocarcinoma distant metastasis remain largely unknown. To preliminary clarify the underlying mechanisms, we investigate the correlation between CCR9 and ALDH1A1 + cancer stem cells (CSCs), as well as the effect of CCR9 on the migration and invasion of CSCs. Methods : Immunohistochemistry was performed to detect the expression of CCR9 in lung adenocarcinoma tissues. The correlations of CCR9 with distant metastasis and overall survival were investigated. Serial paraffin-embedded tissue blocks were used to detect ALDH1A1 + CSCs expression. The correlations between CCR9 expression and ALDH1A1 + CSCs were evaluated. We further studied the effect of CCR9/CCL25 on the migration and invasion of CSCs using transwell assays. Results : There were positive correlations between CCR9 expression and distant metastasis, as well as poor overall survival. Patients with high CCR9 expression were more likely to develop distant metastasis and demonstrated poorer overall survival than patients with low CCR9 expression. In addition, there was positive correlation between the expression of CCR9 and ALDH1A1 in the same tumor microenvironment. ALDH high CSCs demonstrated enhanced expression of CCR9 than ALDH low cells. Further transwell assays demonstrated that the numbers of CSCs migrated or invaded in response to CCL25 were more than that without CCL25 stimulation. Additional application of anti-CCR9 antibody reversed the CCL25-induced migration and invasion of CSCs. Conclusions : In summary, our study demonstrated that CCR9/CCL25 promoted the migration and invasion of CSCs, which might contribute to distant metastasis and poor overall survival. Our findings provided evidence that CCR9/CCL25 could be used as novel therapeutic targets for lung adenocarcinoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Aldehyde dehydrogenases catalyze the NAD(P) + -dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower K m values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of k cat /K m . The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In breast cancer (BC), the presence of cancer stem cells (CSCs) has been related to relapse, metastasis, and radioresistance. Radiotherapy (RT) is an extended BC treatment, but is not always effective. CSCs have several mechanisms of radioresistance in place, and some miRNAs are involved in the cellular response to ionizing radiation (IR). Here, we studied how IR affects the expression of miRNAs related to stemness in different molecular BC subtypes. Exposition of BC cells to radiation doses of 2, 4, or 6 Gy affected their phenotype, functional characteristics, pluripotency gene expression, and in vivo tumorigenic capacity. This held true for various molecular subtypes of BC cells (classified by ER, PR and HER-2 status), and for BC cells either plated in monolayer, or being in suspension as mammospheres. However, the effect of IR on the expression of eight stemness- and radioresistance-related miRNAs (miR-210, miR-10b, miR-182, miR-142, miR-221, miR-21, miR-93, miR-15b) varied, depending on cell line subpopulation and clinicopathological features of BC patients. Therefore, clinicopathological features and, potentially also, chemotherapy regimen should be both taken into consideration, for determining a potential miRNA signature by liquid biopsy in BC patients treated with RT. Personalized and precision RT dosage regimes could improve the prognosis, treatment, and survival of BC patients., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Epithelial ovarian cancer (EOC) is the most lethal of gynecologic malignancies. The standard-of-care treatment for EOC is platinum-based chemotherapy such as cisplatin. Platinum-based chemotherapy induces cellular senescence. Notably, therapy-induced senescence contributes to chemoresistance by inducing cancer stem-like cells (CSC). However, therapeutic approaches targeting senescence-associated CSCs remain to be explored. Here, we show that nicotinamide phosphoribosyltransferase (NAMPT) inhibition suppresses senescence-associated CSCs induced by platinum-based chemotherapy in EOC. Clinically applicable NAMPT inhibitors suppressed the outgrowth of cisplatin-treated EOC cells both in vitro and in vivo . Moreover, a combination of the NAMPT inhibitor FK866 and cisplatin improved the survival of EOC-bearing mice. These phenotypes correlated with inhibition of the CSCs signature, which consists of elevated expression of ALDH1A1 and stem-related genes, high aldehyde dehydrogenase activity, and CD133 positivity. Mechanistically, NAMPT regulates EOC CSCs in a paracrine manner through the senescence-associated secretory phenotype. Our results suggest that targeting NAMPT using clinically applicable NAMPT inhibitors, such as FK866, in conjunction with platinum-based chemotherapy represents a promising therapeutic strategy by suppressing therapy-induced senescence-associated CSCs. SIGNIFICANCE: This study highlights the importance of NAMPT-mediated NAD + biosynthesis in the production of cisplatin-induced senescence-associated cancer stem cells, as well as tumor relapse after cisplatin treatment., (©2019 American Association for Cancer Research.)

Cancer stem cells are responsible for breast cancer initiation, metastasis, and relapse. Targeting breast cancer stem cells (BCSCs) using phytochemicals is a good strategy for the treatment of cancer. A silica gel, a reversed-phase C18 column (ODS), a Sephadex LH-20 gel, thin layer chromatography, and high-performance liquid chromatography (HPLC) were used for compound isolation from Saururus chinensis extracts. The isolated compound was identified as machilin D by mass spectrometry and nuclear magnetic resonance (NMR). Machilin D inhibited the growth and mammosphere formation of breast cancer cells and inhibited tumor growth in a xenograft mouse model. Machilin D reduced the proportions of CD44 + /CD24 - and aldehyde dehydrogenase 1 (ALDH1)-positive cells. Furthermore, this compound reduced the nuclear localization of the NF-κB protein and decreased the IL-6 and IL-8 secretion in mammospheres. These results suggest that machilin D blocks IL-6 and IL-8 signaling and induces CSC death and thus may be a potential agent targeting BCSCs., Competing Interests: The authors declare no conflict of interest.

Poor chemotherapy response remains a major treatment challenge for high-grade serous ovarian cancer (HGSC). Cancer stem cells are the major contributors to relapse and treatment failure as they can survive conventional therapy. Our objectives were to characterise stemness features in primary patient-derived cell lines, correlate stemness markers with clinical outcome and test the response of our cells to both conventional and exploratory drugs. Tissue and ascites samples, treatment-naive and/or after neoadjuvant chemotherapy, were prospectively collected. Primary cancer cells, cultured under conditions favouring either adherent or spheroid growth, were tested for stemness markers; the same markers were analysed in tissue and correlated with chemotherapy response and survival. Drug sensitivity and resistance testing was performed with 306 oncology compounds. Spheroid growth condition HGSC cells showed increased stemness marker expression (including aldehyde dehydrogenase isoform I; ALDH1A1) as compared with adherent growth condition cells, and increased resistance to platinum and taxane. A set of eight stemness markers separated treatment-naive tumours into two clusters and identified a distinct subgroup of HGSC with enriched stemness features. Expression of ALDH1A1, but not most other stemness markers, was increased after neoadjuvant chemotherapy and its expression in treatment-naive tumours correlated with chemoresistance and reduced survival. In drug sensitivity and resistance testing, five compounds, including two PI3K-mTOR inhibitors, demonstrated significant activity in both cell culture conditions. Thirteen compounds, including EGFR, PI3K-mTOR and aurora kinase inhibitors, were more toxic to spheroid cells than adherent cells. Our results identify stemness markers in HGSC that are associated with a decreased response to conventional chemotherapy and reduced survival if expressed by treatment-naive tumours. EGFR, mTOR-PI3K and aurora kinase inhibitors are candidates for targeting this cell population. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Cancer stem cells (CSCs) play a key role in carcinogenesis and progression of head and neck squamous cell carcinomas (HNSCC). The most common markers indicating for CSCs are: CD44, CD24, CD133, ALDH1A1. Our objective was to evaluate the prognostic potential of CSC markers in HNSCC. The study included 49 patients treated for primary HNSCC, 11 patients with upper respiratory tract epithelial dysplasia and 12 subjects with the normal pharyngeal mucosa as a control group. The frequency and expression levels of the four CSC markers were assessed by immunohistochemistry. Univariate and multivariate analyses were used to correlate CSC expression levels with tumor stage, lymph node metastases or overall survival (OS). CD44, CD24, CD133, ALDH1A1 were widely expressed in tumors, whereas CD44 was found to be higher in cancer tissue (P = 0.001). ALDH1A1 expression levels were found to be significantly higher in T3-T4 tumors vs. T1-T2 tumors (P = 0.05). Lymph node metastases had significantly higher expression levels of CD24 (P = 0.01) and CD133 (P < 0.05) than primary tumors. Multifactorial analysis revealed that overall survival (OS) for patients with ALDH1A1 negative tumors was 5.25 times higher than for patients with ALDH1A1 positive (ALDH1A1+) tumors (P = 0.01). On univariate and multivariate analysis, only ALDH1A1 positivity had a significant effect on OS of HNSCC patients (HR = 2.47 for P = 0.02). Immunohistochemistry-based assessments of CSC marker expression in HNSCC has significant predictive implications for patients with HNSCC. The frequency of CSCs in the tumor, specifically of ALDH1A1+ cells correlated with five-year OS in these patients., (Copyright © 2019. Published by Elsevier B.V.)

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.

The correlation of ALDH1 and Bmi1 expression in potentially malignant oral erythroplakia (OE) with oral carcinoma development was reported in our earlier study. Interestingly, a model of field cancerization orchestrated by the cancer stem cells (CSC) was proposed and suggested the identification of CSC-specific markers is useful for prognosis and providing novel targets for prevention and treatment of field cancerization. We revisited the correlation of ALDH1 and Bmi1 expression in OE with the second and multiple carcinomas development. Strikingly, we observed that the expression of ALDH1 and Bmi1 within a single potentially malignant OE lesion significantly correlate with subsequently developing multiple and multifocal carcinomas, which parallels the process of oral field cancerization. Significantly, ALDH1 and Bmi1 are well-defined markers of CSC for head and neck cancer. Consequently, we provided a preliminary evidence for CSC driving the process of field cancerization., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Sorafenib, a small-molecule tyrosine kinase inhibitor with antiangiogenic activity, has been used in liver cancer and kidney cancer treatments. However, clinical trials with sorafenib for breast cancer were stopped in phase III due to limited efficacy. The existence of heterogeneous vasculatures involving tumor cells, such as vessel-like structures formed by vasculogenic mimicry and mosaic vessels, and their resistance to antiangiogenic therapy are thought to be a possible reason for failure of sorafenib therapy. Nevertheless, the features and mechanism of vasculogenesis by tumor cells remain unclear. In the present study, we found that breast cancer stem-like cells (BCSLCs, ALDH1+ cells) were involved in vasculogenic mimicry and mosaic vessel formation in triple-negative breast cancer tissues. Further, only ALDH1+ BCSLCs sorted from MDA-MB-231 could exhibit the tube formation and angiogenesis ability. Sorafenib could inhibit vascularization from endothelial cells rather than that from ALDH1+ cells. α-SMA was identified as a key molecule in vascular formation of BCSLCs. Mechanistically, HIF-1α enhanced the mRNA and protein levels of α-SMA by binding to the HRE element in the promoter directly and meanwhile increased the BCSLCs population. Interestingly, pigment epithelium-derived factor (PEDF), an endogenous angiogenesis inhibitor, could inhibit both endothelial cell-derived and tumor cell-derived angiogenesis by downregulating HIF-1α in breast cancer. Our finding clarified the possible reason for the poor outcome of anti-angiogenesis therapy and PEDF may have the therapeutic potential.

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.

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.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and treatment-resistant malignancy. The lack of pathway-informed biomarkers hampers the development of rational diagnostics or therapies. Recently, the protein abnormal spindle-like microcephaly-associated (ASPM) was identified as a novel Wnt and stemness regulator in PDAC, while the pathogenic roles of its protein isoforms remain unclarified. We developed novel isoform-specific antibodies and genetic knockdown (KD) of putative ASPM isoforms, whereby we uncovered that the levels of ASPM isoform 1 (iI) and ASPM-iII are variably upregulated in PDAC cells. ASPM isoforms show remarkably different subcellular locations; specifically, ASPM-iI is exclusively localized to the cortical cytoplasm of PDAC cells, while ASPM-iII is predominantly expressed in cell nuclei. Mechanistically, ASPM-iI co-localizes with disheveled-2 and active β-catenin as well as the stemness marker aldehyde dehydrogenase-1 (ALDH-1), and its expression is indispensable for the Wnt activity, stemness, and the tumorigenicity of PDAC cells. By contrast, ASPM-iII selectively regulates the expression level of cyclin E and cell cycle progression in PDAC cells. The expression of ASPM-iI and ASPM-iII displays considerable intratumoral heterogeneity in PDAC tissues and only that of ASPM-iI was prognostically significant; it outperformed ALDH-1 staining and clinico-pathological variables in a multivariant analysis. Collectively, the distinct expression patterns and biological functions of ASPM isoforms may illuminate novel molecular mechanisms and prognosticators in PDAC and may pave the way for the development of therapies targeting this novel oncoprotein. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)