Aldefluor Assay Research Articles

Radiation-Induced Bystander Effects of Adipose-Derived Mesenchymal Stem Cells.

ObjectiveThe interaction of tumor cells with surrounding stem cells such as adipose-derived mesenchymal stem cells (ASCs) would be a crucial mechanism of tumor progression. It has been shown that irradiation can affect tumor microenvironment through different mechanisms. Given that, we aimed to examine the bystander radiation-induced effects of ASCs on different cancer cell lines.Materials and MethodsIn this experimental study, ASCs were extracted from five healthy donors, cultured and then irradiated with a 5Gy of gamma radiation. Following 72 hours of incubation, irradiated ASCs-conditioned media (IACM) and non-irradiated ASCs-conditioned media (NIACM) were collected. Following incubation of different cell lines, Jurkat, LNCaP, U87-MG, MDA-MB-231 and MCF-7, in different media, DMEM, NIACM, and IACM, ALDEFLUOR assay and wound healing assays, were conducted. Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression of ATP-binding cassette transporter genes, ABCA1 and ABCG2, was measured in these cell lines. ResultsNIACM significantly increased ALDH activity in MDA-MB-231 cell (P=0.02), while IACM was associated with significant decrease in the LNCaP and MCF-7 cell lines, respectively P=0.02, P=0.03, compared to DMEM as the control. The area of the scratch site was significantly reduced in MDA-MB-231 cells cultured with NIACM compared to DMEM (P=0.04). Furthermore, ABCA1 mRNA expression was considerably decreased in IACM- but not in DMEMtreated LNCaP line (P=0.01).ConclusionIt seems, after exposing to radiation, ASCs modify to prevent tumor development and metastasis through their radiation-induced bystander effects. Therefore, a better understanding of ASCs function in the tumor microenvironment may provide new insights into therapeutic strategies to surmount radio-resistance in cancer treatment.

LIN28B Enhanced STAT3 Signaling Regulates Inflammatory Response and Chemotherapeutic Resistance in Cholangiocytes.

Background:LIN28B is functionally driving malignant transformation and relevance to the worse disease outcomes by promoting cancer aggressiveness. However, a typical role of LIN28B in cholangiocarcinoma (CCA) is primarily unknown. In this study, the tumorigenic potential of LIN28B in the cholangiocyte context was investigated. Methods: Stable LIN28B expression in MMNK-1 cells was generated by infecting with retrovirus-containing LIN28B gene. LIN28B-overexpressing cells were further validated the amount of released cytokines by using human cytokine arrays. After treatment of chemo-drugs, cell viability was subsequently measured using MTT assay. Aldehyde dehydrogenase (ALDH) activity was determined using ALDEFLUOR assay Kit and analyzed by flow cytometry. The mRNA and protein expression levels were respectively assayed by RT-qPCR and western blot. Results:Cytokine release results showed that numerous inflammatory cytokines-chemokines related to cancer initiation and development, such as IL-8, IL-6, VEGF, MCP1, TNF-α were significantly increased in LIN28B-overexpressing MMNK-1 cells. Drug sensitivity test showed that LIN28B-overexpressing MMNK-1 treated cells had a high percentage of cell viability compared to MMNK-1-control treated cells. Activity and expression level of a cancer stem cell marker, ALDH was significantly elevated in LIN28B-overexpressing MMNK-1 cells. Moreover, the activation of an oncogenic signaling pathway, signal transducer and activator of transcription 3 (STAT3) was enhanced in LIN28B-overexpressing MMNK-1 cells. Whereas, growth capacity of LIN28B-overexpressing MMNK-1 cells was found to be reduced in STAT3 inhibition. Conclusion:LIN28B can regulate the inflammatory response and resistance to chemotherapy of cholangiocytes through modulation of STAT3 signaling pathway.A recent study suggests that activated cholangiocytes can be induced by regulation of LIN28B/STAT3 pathway and this may partially contribute to the initiating CCA. Here, LIN28B and its downstream signaling could be considered as an attractive therapeutic target in patients with CCA.

A direct negative feedback loop of miR-4721/FOXA1/Nanog promotes nasopharyngeal cell stem cell enrichment and metastasis

ObjectiveThe recurrence and metastasis of nasopharyngeal cancer (NPC) may be mainly attributed to the persistence of cancer stem cells (CSCs); however, the linkage mechanism has yet to be fully elucidated.MethodsThe levels of miR-4721, FOXA1, and Nanog expression in NPC were detected by in situ hybridization and immunohistochemistry. In vivo and in vitro metastasis assays confirmed miR-4721 promotes cell migration and invasion. Tumor spheroid formation assay, side population (SP) assay, and ALDEFLUOR assay verified miR-4721 regulates cancer stem cell-like properties. Luciferase reporter assay showed that miR-4721 directly regulates FOXA1 and FOXA1 effects the promoter activity of miR-4721 and Nanog. Chromatin immunoprecipitation (ChIP) analysis and electrophoresis mobility shift assay (EMSA) revealed that FOXA1 combined the promoter region of human miR-4721 and Nanog and the possible mechanism was also analyzed.ResultsIn this study, a new mechanism of NPC tumorigenesis related to miR-4721 was verified. We found that miR-4721, FOXA1 and Nanog control their expressions through a negative feedback loop and then activate the downstream regulator of stem cell signaling to promote the enrichment and metastasis of NPC stem cells.ConclusionThese findings elucidate that the feedback loop of miR-4721/FOXA1/Nanog can regulate stemness and metastasis in NPC and may provide an experimental theoretical basis for metastasis and treatment resistance in NPC.

GRIK2 is a target for bladder cancer stem-like cell-targeting immunotherapy.

Recent studies have revealed that treatment-resistant cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be targeted by cytotoxic T lymphocytes (CTLs). CTLs recognize antigenic peptides derived from tumor-associated antigens; thus, the identification of tumor-associated antigens expressed by CSCs/CICs is essential. Human leucocyte antigen (HLA) ligandome analysis using mass spectrometry enables the analysis of naturally expressed antigenic peptides; however, HLA ligandome analysis requires a large number of cells and is challenging for CSCs/CICs. In this study, we established a novel bladder CSC/CIC model from a bladder cancer cell line (UM-UC-3 cells) using an ALDEFLUOR assay. CSCs/CICs were isolated as aldehyde dehydrogenase (ALDH)-high cells and several ALDHhigh clone cells were established. ALDHhigh clone cells were enriched with CSCs/CICs by sphere formation and tumorigenicity in immunodeficient mice. HLA ligandome analysis and cap analysis of gene expression using ALDHhigh clone cells revealed a distinctive antigenic peptide repertoire in bladder CSCs/CICs, and we found that a glutamate receptor, ionotropic, kainite 2 (GRIK2)-derived antigenic peptide (LMYDAVHVV) was specifically expressed by CSCs/CICs. A GRIK2 peptide-specific CTL clone recognized GRIK2-overexpressing UM-UC-3 cells and ALDHhigh clone cells, indicating that GRIK2 peptide can be a novel target for bladder CSC/CIC-targeting immunotherapy.

Abstract 1110: Targeting β-catenin enhances the therapeutic efficacy of osimertinib in EGFR- mutant non-small cell lung cancer (NSCLC)

Abstract In the US, ~15% of patients with metastatic lung adenocarcinoma have tumors harboring “driver” mutations in the EGFR gene, which demonstrate major clinical responses to EGFR Tyrosine Kinase Inhibitors (TKIs). The third-generation EGFR TKI osimertinib shows superior clinical activity compared to early-generation EGFR TKIs, however, essentially all tumors eventually develop resistance to osimertinib. We previously demonstrated that EGFR TKIs induce “adaptive persistence” in cancer cells with stem-like properties, allowing these cells to survive and acquire resistance. Activation of β-catenin and downstream signaling components is one of the major pathways enabling this adaptive persistence. Our goal is to identify and validate β-catenin inhibitors that have the potential to improve the depth and duration of response to osimertinib in EGFR-mutant NSCLC. We used the EGFR-mutant NSCLC cell lines HCC827, HCC4006, and H1975 to evaluate the effects of treatment with osimertinib and a β-catenin inhibitor, BC2059. Cell viability assays were done to determine the IC50 values of BC2059 in this panel of cell lines. ALDEFLUOR assays were performed to assay for induced stem-ness. We also assessed β-catenin protein levels and downstream signaling events in various treatment conditions. To evaluate the preclinical activity of β-catenin inhibition, we treated mice bearing HCC4006 and HCC827 xenografts with osimertinib alone or in combination with BC2059. Preclinical activity was assessed through progression free survival and overall survival. Our in vitro studies demonstrated that NSCLC cell lines are sensitive to β-catenin inhibition in a time and dose-dependent manner, and the addition of BC2059 to osimertinib enhances tumor cell killing. Osimertinib treatment increased non-phosphorylated and total β-catenin levels in EGFR-mutant cell lines. We further showed that osimertinib treatment induced ALDH activity that was sensitive to β-catenin inhibition in HCC4006 and HCC827 cells. BC2059 decreased osimertinib-induced ALDH positivity by >10 fold in HCC4006 and ~2 fold in HCC827 cells. Osimertinib treatment increased levels of the secreted β-catenin transcriptional target PAI1 in cultured media, which was inhibited by BC2059. Our preclinical studies also demonstrated that the combination of osimertinib and BC2059 decreased tumor recurrence and improved overall survival in mice bearing EGFR-mutant human lung cancer xenografts. Overall, our studies show that osimertinib in combination with the β-catenin inhibitor BC2059 enhances osimertinib efficacy and delays tumor recurrence, possibly by targeting drug-tolerant persistent clones that are enriched for with EGFR TKI treatment alone. These data provide strong rationale for a clinical trial that evaluates the combination of osimertinib and BC2059 in patients with metastatic EGFR-mutant NSCLC. Citation Format: Shankar Suman, Shu-Xiao Guan, Nastaran Navari, Rajeswara Rao Arasada, David P. Carbone, Regan M. Memmott. Targeting β-catenin enhances the therapeutic efficacy of osimertinib in EGFR- mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1110.

A novel role of ADGRF1 (GPR110) in promoting cellular quiescence and chemoresistance in human epidermal growth factor receptor 2‐positive breast cancer

Purpose Adhesion G protein coupled receptors (aGPCRs) remains least explored GPCRs family for their role in health and disease. The aGPCR, ADGRF1 (GPR110), is overexpressed and predicts poor survival in various cancer types. However, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We have reported that ADGRF1 is overexpressed in tumorigenic population and in anti-HER2 therapy-resistant derivatives of various HER2+ breast cancer (BC) cell line models. Further, we have reported that ADGRF1 knockdown reduces tumorigenesis and metastasis in HER2+ BC cells. In this study, we evaluated the effects of ADGRF1 overexpression (OE) on tumorigenesis, signaling pathways, and outcomes in HER2+ BC. Methods Stable clones of 2 HER2+ BC cell lines, BT474 and SKBR3, were generated to overexpress ADGRF1 construct in doxycycline-inducible manner. The effect of ADGRF1 on tumorigenesis was evaluated in vitro using soft agar assays, mammosphere assay, and Aldefluor assay. The effect on the HER pathway was evaluated by assessing expression of phospho- and total-HER1 and -HER2 and potency of anti-HER2 drugs. G protein coupling of ADGRF1 was determined by co-immunoprecipitation of Gαs and Gαq and detecting second messengers, cAMP and IP1, in presence/absence of synaptamide, an ADGRF1 agonist. Downstream pathways of ADGRF1 were explored by RNAseq and Reverse Phase Protein Arrays (RPPA) and functional assays. ADGRF1 expression was assessed in BC patients from The Cancer Genome Atlas. ADGRF1 OE effects on survival was evaluated using the Molecular Taxonomy of Breast Cancer International Consortium dataset. Results ADGRF1 OE enhanced the number of colonies and mammospheres and increased Aldefluor+ tumorigenic cell population, suggesting a role in tumorigenesis. ADGRF1 OE did not alter the expression total- or phospho-HER1/-HER2 proteins or efficacy of anti-HER2 drugs. Overexpressed ADGRF1 co-immunoprecipitated with Gαs and Gαq proteins and increased cAMP and IP1. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 OE, suggesting pro-tumorigenic effects of the ADGRF1-Gαs pathway. Synaptamide, did not alter cAMP or IP1 with/without ADGRF1 OE or the number of mammospheres. Integration of RNA-Seq and RPPA data showed that ADGRF1 OE inhibited cell cycle. We detected G0/1 arrest by cell cycle analysis with ADGRF1 OE. The protein level of the proliferation marker Ki67 was reduced upon ADGRF1 OE. Also, ADGRF1 OE led to a 10-fold reduction in potency of docetaxel, a chemotherapy drug used in HER2+ BC. ADGRF1 was overexpressed and amplified in basal and HER2 compared to luminal A and B BCs. High ADGRF1 expression predicted poor survival in HER2 subtype, but not in basal BC. Conclusions: ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.

Autophagy Regulates Cancer Stem Cell Properties in Triple Negative Breast Cancer Via miR-181a-Mediated Regulation of ATG5/ATG2B

Background: Cancer stemness, miRNA, and autophagy flux are known to contribute to the maintenance of cancers; however, determination of the precise interactions among autophagy, miRNAs, and cancer stemness requires further investigation. Methods: Western blot, immunocytochemistry, immunofluorescence staining, quantitative real-time PCR were performed to detect gene expression. Tumorsphere formation assay, Aldefluor assay, limiting dilution assay were carried out to measure cancer stemness. Acridine orange and Cyto-ID® staining were used to detect autophagy vacuoles. Findings: Autophagy flux was inhibited in triple-negative breast cancer (TNBC) cancer stem cells (CSCs). Moreover, miRNA-181a (miR-181a) is upregulated both in TNBC CSCs and patients. ATG5 and ATG2B participate in the early formation of autophagosomes and were revealed as targets of miR-181a. Inhibition of miR-181a expression led to attenuation of cancer stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. Interpretation: It could be suggested that luminal and triple-negative breast cancer require distinct therapeutic approaches because of their different expressions of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. Autophagy inhibits cancer stemness through the miR-181a-regulated mechanism in TNBC. Promoting tumor-suppressive autophagy using curcumin may be a potential method for the treatment of TNBC. Funding: This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (2019R1A2B5B03069738, NRF- 2016R1A5A1011974). Declaration of Interest: All the authors declared no competing interests. Ethical Approval: This study was approved by the 235 Institutional Review Board of Seoul National University Hospital (IRB number: 1704-236 019-843). A breast cancer tissue array (BC081120f, US Biomax Inc, USA).

Tamoxifen induces stem-like phenotypes and multidrug resistance by altering epigenetic regulators in ERα+ breast cancer cells.

To understand the mechanism underlying tamoxifen-induced multidrug resistance (MDR) and stem-like phenotypes in breast cancer cells, we treated the MCF-7 cells with 4-hydroxy-tamoxifen (TAM) for 6 months continuously and established MCF-7 tamoxifen resistance (TR) phenotypes. In the present study, the following methods were used: cell viability assay, colony formation, cell cycle analysis, ALDEFLUOR assay, mammosphere formation assay, chromatin immunoprecipitation (ChIP) assay, PCR array, western blot analysis and quantitative reverse transcription polymerase chain reaction (QRT-PCR). The expression of ERα was significantly higher in MCF7-TR cells when compared with parental MCF-7 cells. MCF7-TR cells exposed to TAM showed a significant increase in the proliferation and rate of colony formation. The number of cancer stem cells was higher in MCF7-TR cells as observed by the increase in the number of ALDH+ cells. Furthermore, the number of mammospheres formed from the FACS-sorted ALDH+ cells was higher in MCF7-TR cells. Using PCR array analysis, we were able to identify that the long-term exposure of TAM leads to alterations in the epigenetic and MDR stem cell marker genes. Furthermore, western blot analysis demonstrated elevated levels of Notch-1 expression in MCF-TR cells compared with MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay revealed that Notch-1 enhanced the cyclin D1 expression significantly in these cells. In addition, we observed that MCF7-TR cells were resistant to doxorubicin but not the MCF-7 cells. In the present study, we conclude that the treatment with tamoxifen induces multiple epigenetic alterations that lead to the development of MDR and stem-like phenotypes in breast cancers. Therefore, our study provides better insights to develop novel treatment regime to control the progression of breast cancer.

Association of ALDH1A1-NEK-2 axis in cisplatin resistance in ovarian cancer cells

Development of acquired resistance to cisplatin (CDDP) is a major obstacle in the treatment of ovarian cancer patients. According to the cancer stem cell (CSC) hypothesis, the recurrence and chemoresistance are presumed to be linked to cancer stem/progenitor cells. Here, we investigated the CSC-like phenotypes and mechanism of chemoresistance in CDDP resistant ovarian cancer cells. A well-established CDDP sensitive ovarian cancer cell line A2780 and its resistant population A2780-Cp were used. We also developed a supra resistant population (SKOV3-Cp) from a naturally CDDP resistant cell line SKOV3. Both resistant/supra resistant cell lines showed significantly higher self-renewal capability than their parental counterparts. They also showed significant resistance to apoptosis and sub-G1 arrest by CDDP treatment. Stem cell marker ALDH1 positivity rates were higher both in A2780-Cp and SKOV3-Cp cell lines than in their counterparts, quantified by Aldefluor assay kit. Hoechst 33342 dye effluxing side populations were increased up to about five folds in A2780-Cp cells and two folds in SKOV3-Cp cells compared to A2780 and SKOV3 cells, respectively. Among major stemness related genes (POU5F1/OCT4, SOX2, NANOG, NES, BMI1, KLF4 and ALDH1A1), ALDH1A1 and KLF4 were significantly overexpressed in both resistant/supra resistant cells. Silencing ALDH1A1 in A2780 and A2780-Cp cells using siRNA greatly reduced the stem cell population and sensitized cells to CDDP. Moreover, silencing of ALDH1A1 reduced the transcript and protein level of its downstream target NEK-2. We also observed the downregulation of ABC transporters (ABCB1/MDR1, ABCG2 and ABCC1/MRP1) either by ALDH1A1 or NEK-2 silencing and upreguation of ABCB1/MDR1 due to the overexpression of NEK-2. Taken together, the present study suggests that stemness gene ALDH1A1 can be involved in CDDP resistance through the upregulation of NEK-2 in ovarian cancer.

Abstract B63: Lysophosphatidic acid as a mediator of ovarian cancer cell stemness

Abstract Epithelial ovarian cancer (OC) is the deadliest gynecologic malignancy. Recurrence of advanced, multidrug-resistant metastatic disease after initially good response to standard lines of chemotherapy occurs in the majority of OC patients. Chemoresistant, recurrent OC is essentially fatal. Disease relapse is due to survival and expansion of a small pool of chemorefractory ovarian cancer stem cells (OCSCs). OC evolution to a platinum-resistant state is associated with aberrant epigenetic modifications, including altered DNA methylation and histone modifications. In addition, OC cells residing in the peritoneal cavity are exposed to a variety of external cues arising from the ascitic microenvironment, including the bioactive molecule lysophosphatidic acid (LPA), which potentially contribute to cell stemness and chemoresistance. LPA in the intraperitoneal fluid of OC patients has been strongly implicated in ovarian carcinogenesis and metastasis through a variety of mechanisms. However, regulation of OC cells by LPA is incompletely understood, and the role of LPA in OCSC acquisition/chemoresistance remains unclear. In the current study, we tested the hypothesis that LPA is a mediator of OCSC promotion and survival. Expression of aldehyde dehydrogenase (ALDH), an accepted functional marker of cancer stem cells, was assessed in a panel of high-grade serous ovarian cancer (HGSOC) cell lines (OVSAHO, OVCAR3, OVCAR5, PEO1, and Kuramochi) using an Aldefluor assay. Treatment of HGSOC cells with increasing (0-80uM, 72h) doses of LPA enriched the ALDH+ population in a cell line-specific dose-dependent manner. In PEO1 and Kuramochi cells, intermediate LPA doses (1-10uM) resulted in a 1.5-2.5-fold increase (P<0.05) of ALDH+ cells (from ∼4% to ∼8% in PEO1 and from ∼4% up to ∼10% in Kuramochi, respectively). However, 80uM LPA treatment was necessary to increase (P<0.05) the percentage ALDH+ cells in OVCAR3 (from ∼17% to 34%) and OVSAHO cells (from ∼10% up to 29%). In accordance with the results of the flow cytometry analysis, cancer stem cell properties after LPA treatment (0-80uM, every 72hr, up to 21 days) were enhanced (P<0.05), based on clonogenic assay. In particular, moderate (10-20uM) LPA treatment increased (P<0.05) cell clonogenic survival of PEO1 and Kuramochi, while a greater (80uM) LPA dose was needed for OVCAR3 and OVSAHO. Furthermore, LPA (0-80uM, 14 days) augmented spheroid formation ability in PEO1 cells (3-4-fold increase; P<0.05). Global transcriptomic and epigenomic assessment of altered genes/pathways induced by LPA are currently being examined to assess stemness-promoting pathways and epigenetic changes, which we believe will identify new biologic insights and potential therapeutic targets to eradicate OCSC in patients. Citation Format: Yuliya Klymenko, Kenneth P. Nephew. Lysophosphatidic acid as a mediator of ovarian cancer cell stemness [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B63.

ALDH1A1+ ovarian cancer stem cells co-expressing surface markers CD24, EPHA1 and CD9 form tumours in vivo

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.

All-trans retinoic acid reduces cancer stem cell-like cell-mediated resistance to gefitinib in NSCLC adenocarcinoma cells

BackgroundThe 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 ALDH1A1bright /CD44high 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.MethodsTreatment 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,ResultsGSCs 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.ConclusionOur findings suggest that combination treatment with ATRA prevents gefitinib-induced enrichment of ALDH1A1bright/CD44high CSCs and enhances gefitinib-induced growth inhibition of NSCLC/ADC cells.

DNA methylation maintains the CLDN1-EPHB6-SLUG axis to enhance chemotherapeutic efficacy and inhibit lung cancer progression

The loss of cancer-cell junctions and escape from the primary-tumor microenvironment are hallmarks of metastasis. A tight-junction protein, Claudin 1 (CLDN1), is a metastasis suppressor in lung adenocarcinoma. However, as a metastasis suppressor, the underlying molecular mechanisms of CLDN1 has not been well studied.Methods: The signaling pathway regulated by CLDN1 was analyzed by Metacore software and validated by immunoblots. The effect of the CLDN1-EPHB6-ERK-SLUG axis on the formation of cancer stem-like cells, drug resistance and metastasis were evaluated by sphere assay, aldefluor assay, flow cytometry, migration assay, cytotoxicity, soft agar assay, immunoprecipitation assay and xenograft experiments. Furthermore, the methylation-specific PCR, pyrosequencing assay, chromatin immunoprecipitation and reporter assay were used to study the epigenetic and RUNX3-mediated CLDN1 transcription. Finally, the molecular signatures of RUNX3/CLDN1/SLUG were used to evaluate the correlation with overall survival by using gene expression omnibus (GEO) data.Results: We demonstrated that CLDN1 repressed cancer progression via a feedback loop of the CLDN1-EPHB6-ERK1/2-SLUG axis, which repressed metastasis, drug resistance, and cancer stemness, indicating that CLDN1 acts as a metastasis suppressor. CLDN1 upregulated the cellular level of EPHB6 and enhanced its activation, resulting in suppression of ERK1/2 signaling. Interestingly, DNA hypermethylation of the CLDN1 promoter abrogated SLUG-mediated suppression of CLDN1 in low-metastatic cancer cells. In contrast, the histone deacetylase inhibitor trichostatin A or vorinostat facilitated CLDN1 expression in high-metastatic cancer cells and thus increased the efficacy of chemotherapy. Combined treatment with cisplatin and trichostatin A or vorinostat had a synergistic effect on cancer-cell death.Conclusions: This study revealed that DNA methylation maintains CLDN1 expression and then represses lung cancer progression via the CLDN1-EPHB6-ERK1/2-SLUG axis. Because CLDN1 enhances the efficacy of chemotherapy, CLDN1 is not only a prognostic marker but a predictive marker for lung adenocarcinoma patients who are good candidates for chemotherapy. Forced CLDN1 expression in low CLDN1-expressing lung adenocarcinoma will increase the chemotherapy response, providing a novel therapeutic strategy.

Alternatively Activated Macrophages Are the Primary Retinoic Acid-Producing Cells in Human Decidua.

In situ production and metabolism of all-trans retinoic acid (RA) in decidual tissue are critically important for endometrial stromal differentiation, embryo implantation, and healthy placentation. However, the cellular source(s) of RA in this tissue has yet to be determined. To identify the primary RA-producing cells in human term decidua, we isolated cells from decidua basalis of delivered placenta and quantified cellular retinal dehydrogenase (RALDH) activity, a major biosynthetic enzyme whose activity determines the synthesis of RA from retinol, using an Aldefluor assay and flow cytometry. RA production in decidual tissue and sorted cell subpopulations was evaluated by liquid chromatography-tandem mass spectrometry. CD14+ cells (macrophages/monocytes) showed > 4-fold higher RALDH activity than stromal cells (CD10+), T cells (CD3+), or non-T lymphocytes (CD3-negative). CD11c+ cells that did not co-express CD14 showed about one-third the RALDH activity of their CD14 co-expressing counterparts. The highest RALDH activity was found in "alternatively activated" M2 macrophages delineated by the simultaneous expression of CD14 and CD163. The greater RA synthesizing capacity of M2 versus CD14+CD163-ve (M1) cells was confirmed by direct quantitation of RA biosynthesis from retinol. RA levels in whole decidua were correlated with M2 cell density but not with stromal cell (CD10+) number, the major cell type comprising the decidua. These results identified M2 monocyte/macrophages as the primary source of RA in human term decidua. This finding may have implications for certain pregnancy complications that are known to be associated with reduced numbers of decidual M2 cells.

High aldehyde dehydrogenase activity does not protect colon cancer cells against TPCS2a-sensi-tized photokilling

Aldehyde dehydrogenases (ALDH) are detoxifying enzymes that are upregulated in cancer stem cells (CSCs) and may cause chemo-and ionizing radiation (IR) therapy resistance. By using the ALDEFLUOR assay, CD133 + human colon cancer cells HT-29, were FACSorted into three populations: ALDHbright, ALDHdim and unsorted (bulk) and treated with chemo-, radio- or photodynamic therapy (PDT) using the clinical relevant photosensitizer disulfo-nated tetraphenyl chlorin (TPCS2a/fimaporfin). Here we show that there is no difference in cytotoxic responses to TPCS2a-PDT in ALHDbright, ALDHdim or bulk cancer cells. Likewise, both 5-FU and oxaliplatin chemotherapy efficacy was not reduced in ALDHbright as compared to ALDHdim cancer cells. However, we found that ALHDbright HT-29 cells are significantly less sensitive to ionizing radiation compared to ALDHdim cells. This study demonstrates that the cytotoxic response to PDT (using TPCS2a as photosensitizer) is independent of ALDH activity in HT-29 cancer cells. Our results further strengthen the use of TPCS2a to target CSCs.

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.

Development of a Retinal-Based Probe for the Profiling of Retinaldehyde Dehydrogenases in Cancer Cells.

Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts.

Abstract GMM-058: CBX2 IDENTIFIED AS DRIVER OF ANOIKIS ESCAPE AND DISSEMINATION IN HIGH GRADE SEROUS OVARIAN CANCER

Abstract The vast majority of high grade serous ovarian carcinomas (HGSOC) are diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in a variety of cancers. In prostate cancer, CBX2 is a driver of metastatic progression. Little is known about the role of CBX2 in HGSOC. Our hypothesis is that CBX2 upregulation promotes advanced HGSOC by promoting a stem-like transcriptional profile and inhibiting anoikis. Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were queried to establish the role of CBX2 in HGSOC. In vitro evaluation of CBX2 occurred in OVCAR4, PEO1, and OVCAR8 HGSOC cell lines. PolyHEMA-coated plates forced cells to grow in suspension and simulated anoikis-escape. Quantitative polymerase chain reaction and immunoblots evaluated CBX2 expression. Small hairpin RNAs (shRNAs) knocked CBX2 down for loss of function studies. To mimic HGSOC progression several culture conditions, including 2D, colony formation, and 3D, spheroid, were examined. Secreted Gaussia luciferase (gLuc) activity was utilized as an indicator of proliferation. Stemness was tested with the Aldefluor assay, measuring aldehyde dehydrogenase activity (ALDH). Using patient tumors derived from the Gynecology Tissue and Fluid Bank (GTFB) and a HGSOC tissue microarray (TMA) with matched primary, metastatic, and lymph nodes, a CBX2 expression profile was established. Student's t-test was used to define statistical significance, with a p value of &amp;lt; 0.05. Analysis of GEO databases established CBX2 is upregulated in HGSOC tumors compared to benign tissues. Analysis of TCGA found CBX2 expression conveyed worse disease-free survival (11.7 vs 17.6 months, Log-rank test p-value &amp;lt; 0.005) and overall survival (34 vs. 44.8 months, Log-rank test p-value &amp;lt;0.005). Examination of primary HGSOC tumors confirmed CBX2 was upregulated at the protein level in HGSOC compared to benign tissue. In vitro, OVCAR4, PEO1, and OVCAR8 cells upregulate CBX2 when grown in suspension compared to adherent conditions. CBX2 knockdown led to a significant inhibition of proliferation in 2D, 3D, and in suspension. Forced suspension promoted increased ALDH3A1 expression and ALDH activity. CBX2 knockdown led to a decrease in both ALDH3A1 expression and ALDH activity. HGSOC cells grown in suspension were found to be more chemoresistant compared cells grown under adherent conditions. Similarly, knockdown of CBX2 sensitized HGSOC cells to cisplatin. Examination of primary tissue in a TMA of matched patient samples revealed CBX2 is expressed in primary and metastatic disease. We conclude that CBX2 directly impacts proliferation and is overexpressed in HGSOC. Our work indicates that CBX2 is an important regulator of stem-ness, which could play a role in anoikis escape, HGSOC dissemination, and chemoresistance, suggesting that CBX2 may be associated with more advanced disease. This exploration expands our understanding of molecular drivers of HGSOC progression and potentially identifies a novel therapeutic target. Citation Format: Lindsay J. Wheeler, Zachary L. Watson, Lubna Qamar, Alexandra McMellen, Tomomi Yamamoto, Kian Behbakht, and Benjamin G. Bitler. CBX2 IDENTIFIED AS DRIVER OF ANOIKIS ESCAPE AND DISSEMINATION IN HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-058.

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.

Transcriptomic Analysis of Breast Cancer Stem Cells and Development of a pALDH1A1:mNeptune Reporter System for Live Tracking

Many solid cancers are hierarchically organized with a small number of cancer stem cells (CSCs) able to regrow a tumor, while their progeny lacks this feature. Breast CSC is known to contribute to therapy resistance. The study of those cells is usually based on their cell-surface markers like CD44high /CD24low/neg or their aldehyde dehydrogenase (ALDH) activity. However, these markers cannot be used to track the dynamics of CSC. Here, a transcriptomic analysis is performed to identify segregating gene expression in CSCs and non-CSCs, sorted by Aldefluor assay. It is observed that among ALDH-associated genes, only ALDH1A1 isoform is increased in CSCs. A CSC reporter system is then developed by using a far red-fluorescent protein (mNeptune) under the control of ALDH1A1 promoter. mNeptune-positive cells exhibit higher sphere-forming capacity, tumor formation, and increased resistance to anticancer therapies. These results indicate that the reporter identifies cells with stemness characteristics. Moreover, live tracking of cells in a microfluidic system reveals a higher extravasation potential of CSCs. Live tracking of non-CSCs under irradiation treatment show, for the first time, live reprogramming of non-CSCs into CSCs. Therefore, the reporter will allow for cell tracking to better understand the implication of CSCs in breast cancer development and recurrence.