ALDHhigh Cells Research Articles

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

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. 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. 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. 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.

Interrogating colorectal cancer metastasis to liver: a search for clinically viable compounds and mechanistic insights in colorectal cancer Patient Derived Organoids

BackgroundApproximately 20–50% of patients presenting with localized colorectal cancer progress to stage IV metastatic disease (mCRC) following initial treatment and this is a major prognostic determinant. Here, we have interrogated a heterogeneous set of primary colorectal cancer (CRC), liver CRC metastases and adjacent liver tissue to identify molecular determinants of the colon to liver spreading. Screening Food and Drug Administration (FDA) approved drugs for their ability to interfere with an identified colon to liver metastasis signature may help filling an unmet therapeutic need.MethodsRNA sequencing of primary colorectal cancer specimens vs adjacent liver tissue vs synchronous and asynchronous liver metastases. Pathways enrichment analyses. The Library of Integrated Network-based Cellular Signatures (LINCS)-based and Connectivity Map (CMAP)-mediated identification of FDA-approved compounds capable to interfere with a 22 gene signature from primary CRC and liver metastases. Testing the identified compounds on CRC-Patient Derived Organoid (PDO) cultures. Microscopy and Fluorescence Activated Cell Sorting (FACS) based analysis of the treated PDOs.ResultsWe have found that liver metastases acquire features of the adjacent liver tissue while partially losing those of the primary tumors they derived from. We have identified a 22-gene signature differentially expressed among primary tumors and metastases and validated in public databases. A pharmacogenomic screening for FDA-approved compounds capable of interfering with this signature has been performed. We have validated some of the identified representative compounds in CRC-Patient Derived Organoid cultures (PDOs) and found that pentoxyfilline and, to a minor extent, dexketoprofen and desloratadine, can variably interfere with number, size and viability of the CRC –PDOs in a patient-specific way. We explored the pentoxifylline mechanism of action and found that pentoxifylline treatment attenuated the 5-FU elicited increase of ALDHhigh cells by attenuating the IL-6 mediated STAT3 (tyr705) phosphorylation.ConclusionsPentoxifylline synergizes with 5-Fluorouracil (5-FU) in attenuating organoid formation. It does so by interfering with an IL-6-STAT3 axis leading to the emergence of chemoresistant ALDHhigh cell subpopulations in 5-FU treated PDOs. A larger cohort of CRC-PDOs will be required to validate and expand on the findings of this proof-of-concept study.

Activated breast stromal fibroblasts exhibit myoepithelial and mammary stem cells features

BackgroundActive breast cancer-associated fibroblasts (CAFs) promote tumor growth and spread, and like tumor cells they are also heterogeneous with various molecular sub-types and different pro-tumorigenic capacities. MethodsWe have used immunoblotting as well as quantitative RT-PCR to assess the expression of various epithelial/mesenchymal as well as stemness markers in breast stromal fibroblasts. Immunofluorescence was utilized to assess the level of different myoepithelial and luminal markers at the cellular level. Flow cytometry allowed to determine the proportion of CD44- and ALDH1-positive breast fibroblasts, while sphere formation assay was used to test the ability of these cells to form mammospheres. ResultsWe have shown here that IL-6-dependent activation of breast and skin fibroblasts promotes mesenchymal-to-epithelial transition and stemness in a STAT3- and p16-dependent manner. Interestingly, most primary CAFs isolated from breast cancer patients exhibited such transition and expressed lower levels of the mesenchymal markers N-cadherin and vimentin as compared to their adjacent normal fibroblasts (TCFs) isolated from the same patients. We have also shown that some CAFs and IL-6-activated fibroblasts express high levels of the myoepithelial markers cytokeratin 14 and CD10. Interestingly, 12 CAFs isolated from breast tumors showed higher proportions of CD24low/CD44high and ALDHhigh cells, compared to their corresponding TCF cells. These CD44high cells have higher abilities to form mammospheres and to enhance cell proliferation of breast cancer cells in a paracrine manner relative to their corresponding CD44low cells. ConclusionTogether, the present findings show novel characteristics of active breast stromal fibroblasts, which exhibit additional myoepithelial/progenitor features.

Aldehyde Dehydrogenase, a Marker of Normal and Malignant Stem Cells, Typifies Mesenchymal Progenitors in Perivascular Niches.

Innate mesenchymal stem cells exhibiting multilineage differentiation and tissue (re)generative-or pathogenic-properties reside in perivascular niches. Subsets of these progenitors are committed to either osteo-, adipo-, or fibrogenesis, suggesting the existence of a developmental organization in blood vessel walls. We evaluated herein the activity of aldehyde dehydrogenase, a family of enzymes catalyzing the oxidation of aldehydes into carboxylic acids and a reported biomarker of normal and malignant stem cells, within human adipose tissue perivascular areas. A progression of ALDHLow to ALDHHigh CD34+ cells was identified in the tunica adventitia. Mesenchymal stem cell potential was confined to ALDHHigh cells, as assessed by proliferation and multilineage differentiation in vitro of cells sorted by flow cytometry with a fluorescent ALDH substrate. RNA sequencing confirmed and validated that ALDHHigh cells have a progenitor cell phenotype and provided evidence that the main isoform in this fraction is ALDH1A1, which was confirmed by immunohistochemistry. This demonstrates that ALDH activity, which marks hematopoietic progenitors and stem cells in diverse malignant tumors, also typifies native, blood vessel resident mesenchymal stem cells.

Abstract P1-05-09: Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method

Abstract Purpose: Immunophenotyping is a useful tool for diagnostic and therapeutic assessment of heterogenous breast cancer cells that depend on external factors and growth conditions. The objective of this study was to characterize the phenotype of patient-derived breast cancer cells (PDBCCs) grown in different culture medium and under different conditions including 2D monolayer and non-adherent 3D spheroid culture by using flow cytometry. Methods: PDBCCs and normal breast cells were harvested from fresh surgical specimens (N=11) of breast tumor and normal tissues. PDBCCs were grown in various culture media under monolayer, spheroid and organoid conditions. Flow cytometry, qRT-PCR, ALDEFLUOR assay, β-galactosidase staining and RNA-seq were performed. Results: PDBCC growth rates were different for each patient case. As compared with full media, A83-01 removal increased EpCAM+CD24+ cells (1.8-fold, p<0.029) and decreased EpCAM-CD44high/+CD24- (0.45-fold, p=0.029). EGF removal increased EpCAM+CD49f− cells (14.1-fold, p=0.029). Removal of both EGF and A83-01 induced senescent cells with high β-galactosidase activity. EpCAM-CD44high/+CD24- cells, ALDHhigh cells, and EpCAMlow/-CD49f+ cells were enriched by 3D spheroids relative to 2D monolayer. 562 genes with 2-fold change and, p-value<0.05 were differentially expressed in 3D spheroids compared to 2D monolayers. Based on public database, many genes associated with poor prognosis in 3D spheroids were identified (Table 1). Table 1. Clinically poor prognosis-associated DEGs were selected from tumor spheroids based on recurrence-free survival analysis using Kaplan-Meier Plotter on database that includes estrogen receptor positive patients. B, coefficient of regression; HR, hazard ratio; CI, confidence interval; ER, estrogen receptor, HER2, human epidermal receptor 2; RFS, recurrent free survival; FC, fold change. Conclusion: PDBCC phenotypes can be modulated by external factors and culture methods. Cancer stem-like properties of PDBCCs can be enriched by 3D spheroids. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for heterogenous breast cancer Clinically poor prognosis-associated DEGs based on recurrence-free survival analysisGenes symbolLog2(FC) P<0.05Prognosis95% CI for Exp(B)HRlowerupperP-valueTop 30 of DEGsUpregulated genesMMP110.90Poor1.611.381.877.5e-10BMP68.85Poor1.21.021.040.024MYCN7.20Poor1.231.041.460.015COL22A17.12Poor1.571.172.110.0026CYP1A1(CP11)7.01Poor1.271.051.530.012PGBD56.78Poor1.221.051.420.011S100P6.70Poor1.491.281.733.3e-07CALML56.24Poor1.271.0810480.0034PPP2R2C6.19Poor1.491.131.970.005PMEPA16.07Poor1.181.011.40.043Downregulated geneFOSB-6.36Poor0.630.520.762e-06LINC00844-6.01Poor0.670.480.930.017DLK2-5.46Poor0.820.710.960.011IL33-5.31Poor0.650.550.773.1e-7SCN2B-5.09Poor0.80.670.950.0099CLDN19-4.80Poor0.660.490.890.0065FOS-4.67Poor0.670.570.798.5e-7EGR1-4.63Poor0.680.580.822.3e-0.5JAM2-4.62Poor0.80.680.950.0092MYH11-4.38Poor0.760.660.890.00045ALPL-4.35Poor0.820.710.960.015 Citation Format: Seungyeon RYU, Hoe Suk Kim, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Wonshik Han. Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-09.

The Detection of Stem-Like Circulating Tumor Cells Could Increase the Clinical Applicability of Liquid Biopsy in Ovarian Cancer.

Stem properties allow circulating tumor cells (CTCs) to survive in the bloodstream and initiate cancer progression. We aimed to assess the numbers of stem-like CTCs in patients with ovarian cancer (OC) before treatment and during first-line chemotherapy (CT). Flow cytometry was performed (Cytoflex S (Beckman Coulter, CA, USA)) using antibodies against CD45; epithelial markers EpCAM and cytokeratin (CK) 8,18; mesenchymal vimentin (vim); and stem-like CD44, CD133 and ALDH. This study included 38 stage I–IV OC patients (median age 66 (Q1–Q3 53–70)). The CK+vim- counts were higher (p = 0.012) and the CD133+ALDHhigh counts were lower (p = 0.010) before treatment in the neoadjuvant CT group than in the adjuvant group. The patients with ascites had more CK+vim- cells before treatment (p = 0.009) and less EpCAM-vim+ cells during treatment (p = 0.018) than the patients without ascites. All the CTC counts did not differ significantly in paired samples. Correlations were found between the CK-vim+ and CD133+ALDHhigh (r = 0.505, p = 0.027) and EpCAM-vim+ and ALDHhigh (r = 0.597, p = 0.004) cells before but not during treatment. Multivariate Cox regression analysis showed that progression-free survival was longer with the presence of surgical treatment (HR 0.06 95% CI 0.01–0.48, p = 0.009) and fewer CD133+ALDHveryhigh cells (HR 1.06 95% CI 1.02–1.12, p = 0.010). Thus, CD133+ALDH+ CTCs have the greatest prognostic potential in OC among the phenotypes studied.

New Perspectives in Different Gene Expression Profiles for Early and Locally Advanced Non-Small Cell Lung Cancer Stem Cells.

IntroductionLung cancer is one of the most common cancers in the world, causing over 1.7 million deaths in 2018. Thus far, no effective treatments against lung cancer for advanced stages have been found. For early stages, although surgery is considered the gold standard treatment, 30–55% of patients develop recurrence within the first 5 years of surgery. Our aim is to assess whether cancer stem cells (CSC) display overexpression of a pool of genes that were previously identified for adenocarcinoma recurrence in patients with early and locally advanced stages of non-small cell lung cancer (NSCLC).MethodsThis cross-sectional study was carried out by harvesting surgical tumor specimens obtained from patients harboring early (I-II) and locally advanced (IIIA) stages of NSCLC. For each patient, cell sorting was performed to identify and isolate the ALDHhigh (CSC) and ALDHlow (cancer cells) populations. The mRNA expressions of 31 recurrence-related genes (target genes) in both ALDHhigh and ALDHlow populations were then assessed and compared.ResultsSurgical specimens were obtained from 22 patients harboring NSCLC. Sixteen (51.6%) out of 31 recurrence-related genes were significantly overexpressed in ALDHhigh cells in the early stages and 9 (29.0%) were overexpressed in the locally advanced stages of NSCLC. Overall, the relative mRNA expressions for these recurrence-related genes were higher in early-stage patients. The average fold change, considering all 31 recurrence-related genes together, was 4.5 (95% CI = 3.1-6.3) in early-stage patients and 1.6 (95% CI = 1.2-2.2) in locally advanced-stage patients.ConclusionsOur study represents the first attempt toward identifying genes associated with recurrence that are overexpressed in cancer stem cells in patients with early and locally advanced stages of NSCLC. This finding may contribute to the identification of new target therapies tailored for NSCLC stages.

ALDHHIGH Population Is Regulated by the AKT/β-Catenin Pathway in a Cervical Cancer Model.

ALDH is an enzyme involved in different cellular processes, including cancer. It has been shown that a cellular subpopulation with high ALDH activity (ALDHHIGH) within a tumor is related to functional capabilities such as stemness, chemoresistance, and tumorigenicity. However, few studies have focused on determining the mechanisms behind ALDH activity within the cells. Previously, our group reported that ALDHHIGH cells have higher tumorigenicity in Cervical Cancer (CC) cell lines. Based on this, we were interested to know the molecular mediators of the ALDHHIGH cells, specifically β-catenin, inasmuch as β-catenin is regulated through different pathways, such as Wnt signaling, and that it acts as a transcriptional co-activator involved in cancer progression. In this work, we show that the increase in ALDHHIGH cell percentage is reverted by β-catenin knockdown. Consistently, upon GSK3-β inactivation, a negative regulator of β-catenin, we observed an increase in ALDHHIGH cells. Additionally, we observed a low percentage of cells positive for Fzd receptor, suggesting that in our model there is a low capacity to respond to Wnt ligands. The analysis of ALDHHIGH cells in a sphere formation model demonstrated the active state of AKT. In accordance with this, impairment of AKT activity not only reduced β-catenin active state, but also the percentage of ALDHHIGH cells. This corroborates that AKT acts upstream of β-catenin, thus affecting the percentage of ALDHHIGH cells. In conclusion, our results show that ALDHHIGH cells are dependent on β-catenin, in spite of the Wnt pathway seems to be dispensable, while AKT emerges as central player supporting a mechanism in this important axis that is not yet well known but its analysis improves our understanding of ALDH activity on CC.

The role of myeloid-derived suppressor cells in increasing cancer stem-like cells and promoting PD-L1 expression in epithelial ovarian cancer.

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.

CD44+/EPCAM+ cells detect a subpopulation of ALDHhigh cells in human non-small cell lung cancer: A chance for targeting cancer stem cells?

Objectives: Several studies demonstrated that aldehyde dehydrogenase (ALDH) and CD44 are the most considered cancer stem cells (CSC) markers. However, a comparison between ALDH high cells and CD44+ cells have been previously described with no significant correlation. Indeed, the aim of the present research is to identify a superficial marker able to match with ALDH high cells population in freshly isolated human lung cancer cells.Materials and Methods: This cross-sectional study analyzed the expression of ALDHhigh/low cells and the positivity for CD44 and epithelium cell adhesion molecule (EPCAM) antigens in surgical lung cancer tissues. The main approach was a cytofluorimetric analysis of ALDH expression and positivity for CD44/EPCAM on primary cell population obtained from 23 patients harboring NSCLC.Results: There was a highly positive correlation between the expressions of ALDHhigh and CD44+/EPCAM+ cells, with a Pearson’s correlation coefficient equal to 0.69 (95% CI 0.39–0.86; P = 0.0002), and Spearman’s correlation coefficient equal to 0.52 (P = 0.0124). The average paired difference between the expression of ALDHhigh and CD44+/EPCAM+ cells was very close to 0, being 0.1% (SD 2.5%); there was no difference between these subpopulations in terms of means (95% CI = –1.0; 1.2%, P = 0.8464). These results highlight a strong similarity between ALDHhigh and CD44+/EPCAM+ cells.Conclusions: Our study is the first attempt which identifies a high correlation between the ALDHhigh and the CD44+/EPCAM+ cells, thus suggesting the possibility to use this superficial marker for future target treatments against lung cancer stem cells.

Cancer Stem Cells of Diffuse Large B Cell Lymphoma Are Not Enriched in the CD45+CD19- cells but in the ALDHhigh Cells.

Although the existence of cancer stem cells (CSCs) has been suggested in diffuse large B cell lymphoma (DLBCL), there is still no definitive marker. CD45+CD19- has been regarded as a potential marker of CSCs in mantle cell lymphoma (MCL). So, we explored the role of CD45+CD19- in DLBCL. However, both CD45+CD19- cells and CD45+CD19+ cells did not generate tumors until more than 100,000 cells were inoculated in NOD/SCID mice, even CD45+CD19+ cells generated more and larger tumors, as well as the soft agar colony formation in vitro; The aldehyde dehydrogenase (ALDH) activity was also identified in this study. Only 1,500 ALDHhigh cells were enough to generate tumors in mice while the same number of ALDH- cells were not. Moreover, both groups formed tumors when more cells were inoculated, but ALDHhigh cells formed more and larger tumors. The similar result was obtained in vitro clonogenicity experiments. OCT4, SOX2, Nanog, and ABCG2 genes did not show any difference in CD45+CD19+, CD45+CD19-, ALDHhigh and ALDH- cells. Taken together, CSCs are not enriched in the CD45+CD19- cells but in the ALDHhigh cells in DLBCL cell lines.

Isolation and Identification of Cancer Stem-Like Cells in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Pilot Study.

Background: Lung cancer stem cells (CSCs) share many characteristics with normal stem cells, such as self-renewal and multipotentiality. High expression of aldehyde dehydrogenase (ALDH) has been detected in many tumors, particularly in the CSC compartment, and it plays an important role in tumor proliferation, metastasis, and drug resistance. CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumors. The aim of this study was to isolate and analyze cancer stem-like cells from surgically removed specimens to compare lung adenocarcinoma (ADENO) and squamous (SQUAMO) cell carcinoma.Methods: The ALDEFLUOR assay was used to identify and sort ALDHhigh and ALDHlow human lung cancer cells following tissue digestion. Fluorescence-activated cell sorting analysis for CD44 was performed with tumor cells. Quantitative real-time PCR was performed to assess the expression of SOX2 and NANOG as stemness markers. ALDH1A1 expression was additionally determined by immunohistochemistry. Anchorage-independent ALDHhigh cell growth was also evaluated. ALDHhigh ADENO and SQUAMO cells were cultured to analyze spheroid formation.Results: All specimens contained 0.5–12.5% ALDHhigh cells with 3.8–18.9% CD44-positive cells. SOX2 and NANOG relative expression in ALDHhigh compared to ALDHlow cells in ADENO and SQUAMO was analyzed and compared between the histotypes. Immunohistochemistry confirmed the presence of ALDH1A1 in the sections. SOX2 and NANOG were expressed at higher levels in the ALDHhigh subpopulation than in the ALDHlow subpopulation only in ADENO cells, and the opposite result was seen in SQUAMO cells. In vitro functional assays demonstrated that ALDHhigh cells exhibited migration capacity with distinct behaviors between ALDHhigh spheres in ADENO vs. SQUAMO samples.Conclusions: Our results highlight the importance of a better characterization of cancer stem-like cells in ADENO and SQUAMO histotypes. This may suggest new differential approaches for prognostic and therapeutic purposes in patients with non-small-cell lung cancer.

Correlating tumor-infiltrating lymphocytes and lung cancer stem cells: a cross-sectional study.

Lung cancer stem cells (LCSCs) are endowed with high aldehyde dehydrogenase (ALDH) expression and play roles in tumor proliferation, metastasis, and drug resistance. Their elusive nature may allow them to escape the immune response by tumor-infiltrating lymphocytes (TILs), which can positively affect the outcome in non-small cell lung cancer (NSCLC) patients. Despite independent investigations on both LCSCs and TILs, the relationship between the two has been very marginally considered. We analyzed whether these two cell types may be related as a prerequisite for novel diagnostic and therapeutic approaches. In this cross-sectional study, NSCLC human surgical specimens from 12 patients were tested by ALDEFLUOR assay to identify ALDHhigh cells. Fluorescence-activated cell sorting (FACS) analyses for CD3+, CD4+, and CD8+ TILs were performed in combination with immunohistochemistry evaluation. Statistically positive correlations were found between ALDH+ and CD8+, and between ALDH+ and CD3+ cells populations; no correlation was found between ALDH+ and CD4+ cells. The expression of CD3+ and CD8+ by cells accounted for 40.1% and 58.7%, respectively, of the variability of ALDH+ cell expression by an R-squared index, which highlights the strong correlation between TILs and LCSCs. Immunohistochemistry revealed 6-25% positive cells. We report a correlation between cytotoxic TILs and LCSCs, which may contribute to the future development of targeted therapies focusing on the different roles of lymphocytes against lung cancer.

ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer.

SummaryUterine endometrial cancer is associated with poor survival outcomes in patients with advanced-stage disease. Here, we developed a three-dimensional cell cultivation method of endometrioid cancer stem-like cells with high aldehyde dehydrogenase (ALDH) activity from clinical specimens. ALDH inhibition synergized with paclitaxel to block cancer proliferation. In the clinical setting, high ALDH1A1 expression was associated with poor survival. A high level of ALDH correlated with an increase of glucose uptake, activation of the glycolytic pathway, and elevation of glucose transporter 1 (GLUT1). Blockade of GLUT1 inhibited characteristics of cancer stem cells. Similarly to ALDH inhibition, GLUT1 inhibition synergized with paclitaxel to block endometrial cancer proliferation. Our data indicated that ALDH-dependent GLUT1 activation and the resulting glycolytic activation are of clinical importance for both prognostic evaluation and therapeutic decision-making in endometrial cancer patients. In addition, the synergistic effects of taxane compounds and ALDH or GLUT1 inhibitors may serve as a new clinical treatment option for endometrial cancer.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

SummaryCancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin−CD90−ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin−CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFβ signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFβ signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFβ/TGFBR1 inhibitors.

PRP‑1 significantly decreases the ALDHhigh cancer stem cell population and regulates the aberrant Wnt/β‑catenin pathway in human chondrosarcoma JJ012cells.

Chondrosarcomas are malignant bone tumors refractory to chemotherapy and radiation treatment; thus, novel therapeutic strategies are required. Proline-rich polypeptide 1 (PRP-1) has previously demonstrated antitumor properties in chondrosarcoma. To further investigate the role of PRP-1 in chondrosarcoma cells, its effects on cancer stem cell (CSC) populations were determined by analyzing aldehyde dehydrogenase (ALDH) activity, an established marker of CSCs, in association with regulation of the Wnt/β-catenin signaling. A significant decrease in ALDHhigh CSCs was observed following treatment of chondrosarcoma JJ012 cells with PRP-1. For RT2 profiler PCR array analysis of Wnt/β-catenin signaling genes, cells were sorted into: i) Bulk JJ012 cells; ii) ALDHhigh cells sorted from untreated JJ012 cells (ALDHhigh-untreated); and iii) ALDHlow cells sorted from PRP-1-treated JJ012 cells (ALDHlow-PRP-1). The expression levels of Wnt/β-catenin signaling genes were determined to be downregulated in the ALDHhigh-untreated cells and upregulated in ALDHlow-PRP-1 cells when compared to the bulk JJ012 cells. Additionally, two important oncogenes involved in this pathway, MMP7 and CCND2, were found to be downregulated in the ALDHlow-PRP-1 cells. Immunocytochemistry demonstrated the localization of β-catenin in the nuclei of the PRP-1-treated cells. Western blotting indicated increased β-catenin expression in the ALDHlow-PRP-1 cells compared with the bulk JJ012 cells. Analysis of the cytoplasmic and nuclear fractions of cells treated with increasing concentrations of PRP-1 and β-catenin nuclear translocation inhibitor CGP57380, suggested the nuclear translocation of β-catenin following PRP-1 treatment. In addition, treatment of JJ012 cells with a specific ALDH inhibitor, diethylaminobenzaldehyde, and PRP-1 resulted in a significant decrease in cytoplasmic β-catenin protein expression. This indicated that ALDH inactivation may be associated with the nuclear translocation of β-catenin. Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway has been previously documented. The findings of the present study support the notion that Wnt/β-catenin activation may serve a differential role in sarcomas, limiting tumor progression in association with decreased CSC activity.

Targeting Endometrial Cancer Stem Cell Activity with Metformin Is Inhibited by Patient-Derived Adipocyte-Secreted Factors.

Advanced endometrial cancer continues to have a poor prognosis, due to limited treatment options, which may be further adversely impacted by obesity. Endometrial cancer stem cells have been reported to drive metastasis, chemotherapy resistance and disease relapse, but have yet to be fully characterised and no specific targeted therapies have been identified. Here, we describe the phenotype and genotype of aldehyde dehydrogenase high (ALDHhigh) and CD133+ve endometrial cancer stem cells and how adipocyte secreted mediators block the inhibitory effect of metformin on endometrial cancer stem cell activity. Ishikawa and Hec-1a cell lines were used to characterise ALDHhigh and CD133+ve endometrial cancer cells using flow cytometry, functional sphere assays and quantitative-Polymerase Chain Reaction. The comparative effect of metformin on endometrial cancer stem cell activity and bulk tumour cell proliferation was determined using an Aldefluor and cytotoxicity assay. The impact of adipocyte secreted mediators on metformin response was established using patient-derived conditioned media. ALDHhigh cells demonstrated greater endometrial cancer stem cell activity than CD133+ve cells and had increased expression of stem cell and epithelial-mesenchymal transition genes. Treatment with 0.5–1 mM metformin reduced the proportion and activity of both endometrial cancer stem cell populations (p ≤ 0.05), without affecting cell viability. This effect was, however, inhibited by exposure to patient-derived adipocyte conditioned media. These results indicate a selective and specific effect of metformin on endometrial cancer stem cell activity, which is blocked by adipocyte secreted mediators. Future studies of metformin as an adjuvant therapy in endometrial cancer should be adequately powered to investigate the influence of body mass on treatment response.

Abstract A65: Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential

Abstract Cancer stem cell-like cells (CSC-LCs) contribute to drug resistance and recurrence of ovarian cancer. Strategies that can eradicate CSC-LCs are expected to substantially improve the outcome of ovarian cancer treatments. We have previously identified a class of thioxodihydroquinazolinone small molecules, which have strong synergistic antitumor activity with platinum drugs, the standard chemotherapeutic agents for ovarian cancer treatment. In the current study, using the activity of aldehyde dehydrogenase (ALDH) as a marker of CSC-LCs, we demonstrated that the combination of thioxodihydroquinazolinone compound 19 with cisplatin is able to deplete ALDH-high CSC-LC population in both established platinum-resistant ovarian cancer cell lines and primary ovarian cancer cells from metastatic ascites of a cisplatin-resistant patient. Compound 19 enhanced the accumulation of intracellular cisplatin in ALDH-high ovarian cancer cells. The combination of compound 19 with cisplatin was also able to reduce the sphere-forming capability of cisplatin-resistant ovarian cancer cells. Using a spheroid-based in vitro metastasis model of ovarian cancer, we demonstrated that the combination of compound 19 with cisplatin prevents spheroid ovarian cancer cells from attaching to substratum and spreading. In vivo, the treatment by the combination of compound 19 with cisplatin significantly reduced tumor burden in a cisplatin-resistant intraperitoneal ovarian cancer xenograft mouse model, as compared to cisplatin-alone treatment. Taken together, our study demonstrated that the thioxodihydroquinazolinone compounds are a new class of agents that in combination with cisplatin are capable of eliminating CSC-LCs in ovarian cancer. Further development of thioxodihydroquinazolinone small molecules may yield a more effective treatment for cisplatin-resistant metastatic ovarian cancer. Citation Format: Jing Ma, Joseph Salamoun, Peter Wipf, Robert Edwards, Bennett Van Houten, Wei Qian. Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A65.

Abstract LB-297: HER2-targeted antibody drug conjugates induce host immunity and target cancer stem cells and the efficacy can be enhanced by anti-PD-L1

Abstract HER2 represents an ideal target for cancer immunotherapy. HER2-targeted antibody drug conjugate (ADC) was tested in immunocompromised (SCID) host, which doesn't allow the evaluation of the ADC-induced immune responses. In addition, no potential impact of ADC on cancer stem cells (CSCs) has been reported. Furthermore, tumor cells with PD-L1 expression may escape immune attack by converting PD-1 positive immune effector cells into anergy. We hypothesized that HER2-targeted ADC could modulate host immune response, target HER2high CSCs, and the efficacy of ADC could be enhanced by anti-PD-L1. To test this hypothesis, we used two immunocompetent murine models: mouse breast tumor D2/F2 and TNBC 4T1 with enforced expression of HER2, D2F2/E2 and HER2-4T1 respectively. Both D2F2/E2 and HER2-4T1 could generate tumor in immunocompetent Balb/c mice, which allowed us to test the efficacy and specificity of HER2-targeted ADC, and to evaluate host immune responses. In D2F2/E2 mouse model, we found that HER2-targeted ADC significantly inhibited D2F2/E2 tumor growth, but not the parental D2F2 tumors, in a dose dependent manner. The specificity was confirmed in HER2-4T1 model because we observed significant suppression of HER2-targted ADC on HER2-4T1 tumor growth, but not on the parental 4T1 tumor. In parallel, we observed that HER2-targeted ADC treatment reduced the number of HER2 positive cells, and ALDHhigh cells. ALDHhigh cells have been identified as cancer stem cells in multiple malignancies. We have characterized the stemness of ALDHhigh 4T1 cells. In the present study, we confirmed that the ALDHhigh sub-population of D2F2/E2 cells are highly tumorigenic compared to ALDHlow cells. Next, we tested and observed that PD-L1 expression increased in D2F2/E2 and HER2-4T1 fresh tumor cells than in cell lines. Anti-PD-L1 mAb significantly augmented the therapeutic efficacy of HER2-targeted ADC in both D2F2/E2 and HER2-4T1 tumor models. In addition, anti-PD-L1 plus HER2-targeted ADC further reduced the number of HER2 positive cells, and ALDHhigh cells, but increased the number of CD3+ and CD19+ tumor infiltrating lymphocytes (TILs). Expanded CD3+ TILs from the tumor subjected to HER2-targeted ADC plus anti-PD-L1 treatment killed tumor cells significantly more than mono-treatment. Expanded CD19+ TILs from tumor subjected to HER2-targeted ADC plus anti-PD-L1 produced more IgG than all the controls. Importantly, the IgG could specifically bind to tumor cells, resulting in the highest cytotoxicity of the tumor cells via ADCC. Together, our data indicate that HER2-targeted ADC could induce significant host immune responses, which was evidenced by the detection of anti-tumor CD3+ and CD19+ TILs. We first show that HER2-targeted ADC could target ALDHhigh cancer stem cells, and such effect was significantly enhanced by anti-PD-L1 administration. Citation Format: Leiming Xia, Lu Wen, Ming Lin, Comer Frank, Hinrichs Mary Jane, Oberst Michael, Coats Steven, Yangyi Bao, Alfred E. Chang, Max Wicha, Qiao Li. HER2-targeted antibody drug conjugates induce host immunity and target cancer stem cells and the efficacy can be enhanced by anti-PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-297.

PO-407 Identification of unique antigens on prostate cancer stem cells for cytotoxic T cell recognition

IntroductionCancer stem cells (CSCs) are more resistant to chemo- and radiation therapy than the bulk of the tumour. CSCs, surviving traditional therapies, possess self-renewal capacity and contribute to tumour re-growth. We are investigating the susceptibility of CSCs to antigen-specific killing by T cells. We generated a dataset incorporating all HLA Class I epitopes of the DU145 prostate cancer cell line. We aim to identify novel CSC epitopes which may serve as T cell targets.Material and methodsWe isolated HLA Class I:peptide complexes from DU145 cells by immunoaffinity precipitation and analysed the epitope sequences by liquid chromatography tandem-mass spectrometry (LC-MS/MS). We selected epitopes in the dataset which showed low gene expression in healthy tissues (www.GTexportal.org) and high predicted HLA-binding affinity (http://tools.immuneepitope.org/mhci/). DU145 CSCs were isolated based on aldehyde dehydrogenase (ALDH) activity using FACS. ALDHhigh and ALDHlow DU145 cells were determined to have CSC or non-CSC characteristics, respectively, by in vivo tumour initiation. Gene expression of the epitopes was measured by qPCR in ALDHhigh and ALDHlow DU145 cells. Protein expression was determined by immunofluorescence. We performed homology modelling of the HLA:peptide interfaces and quantified the binding interactions using PISA interface analysis software. We measured T cell activation by selected peptides analysing cytokine release by flow cytometry.Results and discussionsWe selected 42 epitopes from the DU145 dataset with low global tissue expression and predicted HLA binding percentile rank <1%. We identified 11 CSC epitopes by qPCR; 6 upregulated in ALDHhigh DU145 cells (e.g. TACSTD2) and 5 abundant in both ALDHhigh and ALDHlow DU145 cells (e.g. XPO1). We identified 3 HLA:epitope complexes for which the PISA P-value indicated high affinity interface interactions. CD8+ T cells that release cytokines in response to one of these epitopes have been identified. The antigen in question is upregulated in the prostate and testis, thus it is a promising T cell target on prostate CSCs.ConclusionWe have identified prostate CSC epitopes from proteins involved in vascular regulation, calcium signalling and nuclear export. This highlights the potential for targeting CSC epitopes from proteins other than those used for CSC isolation and maybe shared with non cancer stem cells. Our ongoing work involves tetramer based isolation of CSC epitope-specific cytotoxic T cells.