OBJECTIVE: Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) 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.
METHODS: Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow 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 ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay.
RESULTS: Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh 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.
CONCLUSIONS: High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

OBJECTIVE: Emerging evidence suggests that osteosarcoma stem cells (OSCs) may be responsible for tumor initiation propagation, recurrence, and resistance to therapy. We set out to evaluate the relationship between the abundance of ALDH1A1 and CD44-positive cells in biopsy and resection samples on disease recurrence and overall survival.
METHODS: A retrospective review of 20 patients, including biopsy and resection samples, was performed at a comprehensive cancer center. Additionally, we queried the publicly available TARGET dataset of osteosarcoma patients.
RESULTS: Neither the percentages of ALDH1A1-positive cells nor CD44-positive cells were significantly associated with overall mortality or disease recurrence in either biopsy or resection samples. Unlike our institutional data, overall survival was significantly correlated to higher ALDH1A1 expression in the TARGET dataset both in univariate and age-adjusted analyses.
CONCLUSIONS: ADLH1 and CD44, potential markers of OSCs, were not found to be reliable clinical immunohistochemical prognostic markers for osteosarcoma patient survival, specifically disease-free survival. Osteosarcoma patients with high ALDH1A1 RNA expression showed improved overall survival in examining a national genomic database of osteosarcoma patients but again no association with disease-free survival. The potential of CD44 and ALDH1A1 as cellular-specific prognostic markers of survival, and as possible molecular targets, may be limited in osteosarcoma.

Ovarian cancer is the most lethal gynaecological cancer and the eighth most common female cancer. The early diagnosis of ovarian cancer remains a clinical problem despite the significant development of technology. Nearly 70% of patients with ovarian cancer are diagnosed with stages III-IV metastatic disease. Reliable diagnostic and prognostic biomarkers are currently lacking. Ovarian cancer recurrence and resistance to chemotherapy pose vital problems and translate into poor outcomes. Cancer stem cells appear to be responsible for tumour recurrence resulting from chemotherapeutic resistance. These cells are also crucial for tumour initiation due to the ability to self-renew, differentiate, avoid immune destruction, and promote inflammation and angiogenesis. Studies have confirmed an association between CSC occurrence and resistance to chemotherapy, subsequent metastases, and cancer relapses. Therefore, the elimination of CSCs appears important for overcoming drug resistance and improving prognoses. This review focuses on the expression of selected ovarian CSC markers, including CD133, CD44, CD24, CD117, and aldehyde dehydrogenase 1, which show potential prognostic significance. Some markers expressed on the surface of CSCs correlate with clinical features and can be used for the diagnosis and prognosis of ovarian cancer. However, due to the heterogeneity and plasticity of CSCs, the determination of specific CSC phenotypes is difficult.

Among the many consecutive theories of cancer, the stem cell theory is currently the most accepted one. Cancer stem cells are located in small niches with specific environment, renew themselves and are believed to be responsible for many recurrences. They can be highlighted with stem cell markers, but often these markers also label tumor cells, and this may represent a phenotypical change associated with prognosis. In this study, we attempted to match tumor outcomes with the expression of the following stem cell markers: ALDH1, AnnexinA1, CD44, CD117, CD166, Nanog and oct-4. Tissue microarray blocks from triple-negative breast cancers were immunostained for the listed markers, and their expression by the majority of tumor cells (diffuse positivity) was correlated with prognosis. Of the 106 tumors investigated, diffuse positivity was seen in 7 (ALDH1), 33 (AnnexinA1), 53 (CD44), 44 (CD117 membranous only), 49 (CD117), 72 (CD166), 19 (Nanog), and 11 (oct-4) cases. With a median follow-up of 83 months, ALDH1 and CD117 expression was associated with DFS, whereas CD44, CD117 and CD166 were associated with OS estimates, based on Kaplan-Meier analyses. In the multivariate Cox proportional hazard models (including the examined markers and clinicopathological data which had a statistical impact in the univariate analysis), the pN category and the lack of ALDH1 expression were independent prognosticators for DFS, and the pN category and diffuse CD44 staining were independent prognosticators for OS. In the multivariate analysis including all of the examined clinicopathological data and markers, only CD117 showed a statistical impact on OS. We failed to demonstrate a prognostic impact for most stem cell markers tested in triple-negative breast cancer, but lack of ALDH1 staining and CD44 expression appears as of prognostic value, requiring further examination in independent studies.

OBJECTIVE: We have reported that p62 (also known as sequestosome 1) is needed for survival/proliferation and tumor formation by aldehyde dehydrogenase 1 (ALDH1) -positive cancer stem cells (CSCs) and that p62high ALDH1A3high expression is associated with a poor prognosis in luminal B breast cancer. However, the association between p62high ALDH1A3high and the benefit from radiotherapy in patients with luminal B breast cancer remains unclear.
METHODS: Datasets from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) were downloaded, and data from p62high ALDH1A3high luminal B patients treated without or with radiotherapy were analyzed by Kaplan-Meier and multivariate Cox regression analyses. We also performed an in vitro tumor sphere formation assay after X-ray irradiation using p62-knockdown ALDH1high luminal B BT-474 cells.
RESULTS: p62high ALDH1A3high patients had poorer clinical outcomes than other luminal B breast cancer patients treated with radiotherapy. The combination of p62 DsiRNA KD and X-ray irradiation suppressed in vitro tumor sphere formation by ALDH1high BT-474 cells. These results suggest that p62 is involved in the reduced effect of X-ray irradiation on ALDH1-positive luminal B breast CSCs.
CONCLUSIONS: p62 and ALDH1A3 may serve as prognostic biomarkers for luminal B breast cancer patients treated with radiotherapy. Additionally, the combination of p62 inhibition and radiotherapy could be useful for targeted strategies against ALDH1-positive luminal B breast CSCs.

Temozolomide (TMZ) is standard treatment for glioblastoma (GBM); nonetheless, resistance and tumor recurrence are still major problems. In addition to its association with recurrent GBM and TMZ resistance, ALDH1A3 has a role in autophagy-dependent ferroptosis activation. In this study, we treated TMZ-resistant LN229 human GBM cells with the ferroptosis inducer RSL3. Remarkably, TMZ-resistant LN229 clones were also resistant to ferroptosis induction, although lipid peroxidation was induced by RSL3. By using Western blotting, we were able to determine that ALDH1A3 was down-regulated in TMZ-resistant LN229 cells. Most intriguingly, the cell viability results showed that only those clones that up-regulated ALDH1A3 after TMZ withdrawal became re-sensitized to ferroptosis induction. The recovery of ALDH1A3 expression appeared to be regulated by EGFR-dependent PI3K pathway activation since Akt was activated only in ALDH1A3 high clones. Blocking the EGFR signaling pathway with the EGFR inhibitor AG1498 decreased the expression of ALDH1A3. These findings shed light on the potential application of RSL3 in the treatment of glioblastoma relapse.

Cholangiocarcinoma (CCA) is the most recurrent malignant tumor found in the biliary system. It originates from the bile duct epithelial cells characterized by easy metastasis, high intermittent rate, and poor prognosis. Acetaldehyde dehydrogenase 1 (ALDH1), a marker of cancer stem cells, the levels of which are particularly elevated in various of malignant tumors. Additionally, the increased ALDH1 levels are closely related to the degree and prognosis of malignant tumors. This study reviewed the mechanisms underlying the changes in ALDH1 levels in CCA.

Pancreatic ductal adenocarcinoma (PDAC) is the cancer with the poorest prognosis. One of the major properties reflecting its poor prognosis is high-grade heterogeneity, which leads to insensitivity to anticancer treatments. Cancer stem cells (CSCs) acquire phenotypic heterogeneity, generating abnormally differentiated cells by asymmetric cell division. However, the detailed mechanism leading to phenotypic heterogeneity is largely unknown. Here, we showed that PDAC patients with co-upregulation of PKCλ and ALDH1A3 had the poorest clinical outcome. PKCλ knockdown by DsiRNA in the ALDH1high population of PDAC MIA-PaCa-2 cells attenuated the asymmetric distribution of the ALDH1A3 protein. To monitor asymmetric cell division of ALDH1A3-positive PDAC CSCs, we established stable Panc-1 PDAC clones expressing ALDH1A3-turboGFP (Panc-1-ALDH1A3-turboGFP cells). In addition to MIA-PaCa-2-ALDH1high cells, turboGFPhigh cells sorted from Panc-1-ALDH1A3-turboGFP cells showed asymmetric cell propagation of ALDH1A3 protein. PKCλ DsiRNA in Panc-1-ALDH1A3-turboGFP cells also attenuated the asymmetric distribution of ALDH1A3 protein. These results suggest that PKCλ regulates the asymmetric cell division of ALDH1A3-positive PDAC CSCs. Furthermore, Panc-1-ALDH1A3-turboGFP cells can be useful for the visualization and monitoring of CSC properties such as asymmetric cell division of ALDH1A3-positive PDAC CSCs in time-lapse imaging.

Cervical cancer which is caused by persistent infection with oncogenic human papillomavirus (HPV), is the third most common cancer. HPV infection causes the progression of the normal cervix to cervical intraepithelial neoplasia (CIN) because it often occurs at the function conversion of the cervical squamous epithelium and columnar epithelium zone, further to invasive carcinoma. The difference in the ALDH1 expression was very significant. With the progression of cervical cancer, reports explained obviously increased nuclear and cytoplasm ALDH1 staining in comparisons of cervical carcinomas and normal cervix (P < 0.0001), cervical carcinomas compared with CIN (P = 0.0002). Therefore, ALDH1 as a stem marker, not only resists cervical cancer but also resists in normal cervix and CIN tissues. Developing an experimental method to discover cervical cancer earlier is feasible. Furthermore, the ALDH1 was expressed in human cervical cancer cell lines (Hela, SiHa, CaSki, HT-3, and C33A) together with western blot and immunocytochemical analysis. ALDH1 plays a significant role in nuclear and cytoplasm staining by immunochemistry in single or clustered HT-3 and C33A cells. However, western blot and immunochemical analysis did not detect ALDH1 in HeLa or CaSki, SiHa cells. We also discovered that there were no remarkable differences in age, tumor size, clinical TNM staging, multiple pelvic lymph node metastasis, or histological staging (p > 0.05) between the ALDH1-positive groups in 100 cervical cancer tissues. But after the control variable age, different ALDH rating survival function contrasted, it can be concluded that the higher ALDH1 scores with the survival of patients with the worse condition.

Cancer stem cells (CSCs) have high tumorigenicity, high metastasis and high resistance to treatment. They are the key factors for the growth, metastasis and drug resistance of malignant tumors, and are also the important reason for the occurrence and recurrence of tumors. Metabolic reprogramming refers to the metabolic changes that occur when tumor cells provide sufficient energy and nutrients for themselves. Metabolic reprogramming plays an important role in regulating the growth and activity of cancer cells and cancer stem cells. In addition, the immune cells or stromal cells in the tumor microenvironment (TME) will change due to the metabolic reprogramming of cancer cells. Summarizing the characteristics and molecular mechanisms of metabolic reprogramming of cancer stem cells will provide new ideas for the comprehensive treatment of malignant tumors. In this review, we summarized the changes of the main metabolic pathways in cancer cells and cancer stem cells.