ALDH Bright Cells Research Articles

Expression Of Aldehyde Dehydrogenase Reflected Diverse Stem Cell Properties In Acute Myeloid Leukemia

Separation of leukemic stem cells (LSC) and residual hematopoietic stem cells (HSC) from the same individual patient with acute myeloid leukemia (AML) is essential for a proper understanding of the leukemic driving mechanisms. We have studied the role of aldehyde dehydrogenase (ALDH) for this purpose and have defined the functional properties of ALDHbright cells in specific subgroups of AML.We have examined the ALDH activity by flow cytometry in bone marrow samples (BM) from 14 healthy donors and 73 patients with de novo AML. The median frequency of cells with high ALDH activity (ALDHbright cells) in the healthy subjects was 1.92% with a range from 0.58 to 3.16%. For patients with AML, the median number of ALDHbright cells was 0.25% with a broad range from 0.004 to 33.57%. Whereas the majority of patients with AML (n = 56) had low frequencies of ALDHbright cells (median 0.11%; range 0.004 – 1.77%; defined as ALDH-low AML), 17 patients had relatively numerous ALDHbright cells (median 9.01; range 3.54 – 33.57%; defined as ALDH-numerous AML). In both groups, ALDHbright cell populations were highly enriched for CD34+CD38- cells. The ALDHbright cells derived from ALDH-low AML did not contain chromosomal and molecular aberrations characteristic of the original leukemia, and were able to induce multi-lineage hematopoiesis in NSG mouse models. Thus, genetically and functionally normal HSC could be successfully isolated in the ALDHbright subset, whereas LSC were enriched in ALDHdimCD34+CD38- subset for patients with ALDH-low AML. For 17 patients with ALDH-numerous AML, the ALDHbright subset was consistently contaminated with LSC. In clinical follow-ups, patients with ALDH-numerous AML showed resistance to induction chemotherapy and were characterized by a very poor long-term outcome that was comparable to patients with high-risk cytogenetic or molecular genetic markers. In four patients with ALDH-numerous AML we demonstrated that the ALDHbrightCD34+CD38- subset contained chemotherapy-resistant clones with repopulating ability. Furthermore, such ALDHbright cells were characterized by a lower cell-cycle activity and an increased resistance to cytarabine in comparison with ALDHdim blasts in in vitro assays.Our data have provided evidence that LSC and residual HSC can be separated using ALDH in patients with low frequencies of ALDHbright cells. In patients with ALDH-numerous AML, the ALDHbright subset is associated with leukemic features both in vitro and in animal models. Thus our data demonstrated the feasibility of appropriate comparisons of LSC versus HSC from the same patient with specific subtypes of AML and the impact of LSC properties on clinical outcome. Disclosures:Buss:Novartis: Travel support Other; Micromet/Amgen: Reimbursements for participation in a clinical study , Reimbursements for participation in a clinical study Other. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Characterization and Clinical Relevance of ALDHbright Populations in Prostate Cancer

High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance.

Depletion of circulating progenitor cells precedes overt diabetes: A substudy from the VA enhanced fitness trial

BackgroundOne theory of aging and disease development is that chronic injury (pathology) results in activation of regenerative processes and initial repair, with overt disease arising only after exhaustion of reparative capability leads to inadequate repair. While depletion of circulating progenitor cells (CPCs) has been noted in diabetes, the degree to which CPC depletion predates and is associated with propensity to develop overt disease is unclear. MethodsThe Enhanced Fitness trial enrolled overweight/obese (body mass index >25) sedentary patients with glucose intolerance but without overt diabetes. Baseline CPCs were measured in 129 patients based on the cell surface markers CD34, CD133, and aldehyde dehydrogenase (ALDH) activity. HgbA1C, fasting insulin and glucose levels, and HOMA calculations were ascertained. ResultsLower counts of early angiogenic CPCs identified as CD34+, CD34+CD133+, and ALDH-bright (ALDHbr) cells were associated with impairments in glucose homeostasis as reflected by HgbA1C, but not fasting insulin, glucose, or HOMA-IR. These associations remained when corrected for age and cardiovascular risk factors. Conclusions/InterpretationThe numbers of CD34+ and ALDHbr CPCs were significantly lower in patients with impaired glucose tolerance. Depletion of reparative capacity as reflected by loss of CPCs may presage overt disease as exemplified in this pre-diabetes model.

Stem cell-like ALDHbright cellular states in EGFR-mutant non-small cell lung cancer: A novel mechanism of acquired resistance to erlotinib targetable with the natural polyphenol silibinin

The enrichment of cancer stem cell (CSC)-like cellular states has not previously been considered to be a causative mechanism in the generalized progression of EGFR-mutant non-small cell lung carcinomas (NSCLC) after an initial response to the EGFR tyrosine kinase inhibitor erlotinib. To explore this possibility, we utilized a pre-clinical model of acquired erlotinib resistance established by growing NSCLC cells containing a TKI-sensitizing EGFR exon 19 deletion (ΔE746-A750) in the continuous presence of high doses of erlotinib. Genome-wide analyses using Agilent 44K Whole Human Genome Arrays were evaluated via bioinformatics analyses through GSEA-based screening of the KEGG pathway database to identify the molecular circuitries that were over-represented in the transcriptomic signatures of erlotinib-refractory cells. The genomic spaces related to erlotinib resistance included a preponderance of cell cycle genes (E2F1, -2, CDC2, -6) and DNA replication-related genes (MCM4, -5, -6, -7), most of which are associated with early lung development and poor prognosis. In addition, metabolic genes such as ALDH1A3 (a candidate marker for lung cancer cells with CSC-like properties) were identified. Thus, we measured the proportion of erlotinib-resistant cells expressing very high levels of aldehyde dehydrogenase (ALDH) activity attributed to ALDH1/3 isoforms. Using flow cytometry and the ALDEFLUOR® reagent, we confirmed that erlotinib-refractory cell populations contained drastically higher percentages (>4500%) of ALDHbright cells than the parental erlotinib-responsive cells. Notably, strong decreases in the percentages of ALDHbright cells were observed following incubation with silibinin, a bioactive flavonolignan that can circumvent erlotinib resistance in vivo. The number of lung cancer spheres was drastically suppressed by silibinin in a dose-dependent manner, thus confirming the ability of this agent to inhibit the self-renewal of erlotinib-refractory CSC-like cells. This report is the first to show that: (1) loss of responsiveness to erlotinib in EGFR-mutant NSCLC can be explained in terms of erlotinib-refractory ALDHbright cells, which have been shown to exhibit stem cell-like properties; and (2) erlotinib-refractory ALDHbright cells are sensitive to the natural agent silibinin. Our findings highlight the benefit of administration of silibinin in combination with EGFR TKIs to target CSCs and minimize the ability of tumor cells to escape cell death in EGFR-mutant NSCLC patients.

Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway

Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg’s theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka’s stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR—the master switch of cellular catabolism and anabolism—in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1–60, and TRA-1–81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44+ and ALDEFLUOR-stained ALDHbright cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep cells. Consistent with the downregulation of AMPK expression, immunoblotting procedures confirmed upregulation of p70S6K and increased phosphorylation of mTOR in Sox2-overexpressing CSC-like cell populations. Using an in vitro model of the de novo generation of CSC-like states through the nuclear reprogramming of an established breast cancer cell line, we reveal that the transcriptional suppression of mTOR repressors is an intrinsic process occurring during the acquisition of CSC-like properties by differentiated populations of luminal-like breast cancer cells. This approach may provide a new path for obtaining information about preventing the appearance of CSCs through the modulation of the AMPK/mTOR pathway.

Characterization of cardiac-resident progenitor cells expressing high aldehyde dehydrogenase activity.

High aldehyde dehydrogenase (ALDH) activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDHbr) cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDHbr cells in the heart has not been evaluated so far. We have characterized ALDHbr cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDHbr. ALDHvery-br cells were more frequent in neonatal hearts than adult. ALDHbr cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDHvery-br cells, intermediate in ALDHbr cells, and lowest in ALDHdim cells. ALDH1A2 expression was highest in ALDHvery-br cells, intermediate in ALDHdim cells, and lowest in ALDHbr cells. ALDH1A3 and ALDH2 expression was detectable in ALDHvery-br and ALDHbr cells, unlike ALDHdim cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDHbr cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDHbr cells, unlike ALDHdim cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α-actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDHbr cells declined with cell passage. In conclusion, the cardiac-derived ALDHbr population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDHbr cells remains to be evaluated.

Application of Biotechnology in Myocardial Regeneration-Tissue Engineering Triad: Cells, Scaffolds, and Signaling Molecules

Application of Biotechnology in Myocardial Regeneration-Tissue Engineering Triad: Cells, Scaffolds, and Signaling Molecules

Enhanced therapeutic effects of a novel oncolytic and anti-angiogenic vaccinia virus against triple-negative breast cancer

Gholami, Sepideh MD; Marano, Andrew A. BA; Chen, Nanhai G. PhD; Frentzen, Alexa PhD; Chen, Chun-Hao MD; Eveno, Clarisse MD; Lou, Emil MD, PhD; Belin, Laurence MD, MPH; Szalay, Aladar A. PhD; Fong, Yuman MD, FACS Author Information

Abstract 3384: High ALDH activity is associated with high tumorigenicity in prostate cancer

Abstract OBJECTIVE: Tumor initiation and progression might be driven by rare populations of cells endowed with stem-properties, and therefore defined as cancer stem cells (CSC). High Aldehyde dehydrogenase (ALDH) activity has been suggested to selectively identify CSC in several tumor types including breast cancer, and, possibly, prostate cancer (PCA). In this study, we investigated presence and potential CSC characteristics of cells with high ALDH activity (ALDH bright) in PCA cell lines, fresh surgical specimens, and primary cultures. MATERIALS AND METHODS: PC3, Du145, VCaP, and LNCaP PCA cell lines were evaluated. Surgical specimens, including Benign Prostate Hyperplasia (BPH) and PCA, were used directly for gene expression studies. Surgical samples were also enzymatically digested for functional analysis and the establishment of primary cultures. ALDH activity was tested using ALDEFLUOR® technology. Cells were sorted from individual cell lines by flow cytometry and evaluated for CSC properties, including spheroid formation ability, clonogenicity, stemness-related gene expression, ALDH specific isoforms expression, and tumorigenicity upon injection in NOD/SCID mice. RESULTS: PCA cell lines and primary cultures displayed heterogeneous ALDH activity and expression of specific ALDH isoforms. Despite a higher expression of Oct4A and Klf4 stemness associated genes, ALDH bright cells isolated from Du145 and PC3 did not show improved spheroid formation or clonogenic capacity, as compared to their dim counterparts. Interestingly, however, ALDH bright cells were associated with a significantly higher tumorigenic capacity in vivo as compared to ALDH low cells. Nevertheless, ALDH bright cells lost their higher tumorigenic capacity following serial “in vivo” passages. Most importantly, a well defined ALDH bright population could also be detected in cells isolated from PCA specimens (n&amp;gt;20). ALDH activity was consistent with a strong expression of several ALDH specific isoforms in PCA tissues. Notably, a significant increase of defined ALDH isoforms such as ALDH1A3 (p=0.001) was detectable in tissues obtained from patients with PCA as compared to BPH or normal specimens. CONCLUSIONS: ALDH bright subsets can be detected in cells isolated from fresh PCA tissue samples, PCA cell lines and primary cultures. These populations appear to be associated with increased “in vivo” tumorigenicity rather than enhanced “in vitro” stem properties in the cell lines investigated. Importantly, expression of ALDH specific isoforms appears to be increased in clinical PCA as compared to BPH and “normal” samples, thereby suggesting a putative role of ALDH in PCA tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3384. doi:1538-7445.AM2012-3384

Abstract 4390: Heat shock protein (HSP) Grp94-targeted combinatorial immunotherapy for pancreatic cancer

Abstract Pancreatic cancer is among the deadliest of cancers as it is rarely curable and the 5-year survival rate of patients with any stage of the disease is approximately 6%. This dismal clinical picture is reflective of the poor response of this disease to chemo- and radio- therapy, as well as to surgery as conventionally used. The unmet clinical needs for patients with pancreatic cancer highlight the urgency to develop effective novel therapies for this disease. To address this need, we have developed a combinatorial immunotherapy which eliminates differentiated pancreatic adenocarcinoma cells and pancreatic cancer initiating cells (CICs). According to the cancer stem cell theory, CICs are responsible for disease recurrence and metastases. Therefore, they have to be eradicated in order to “cure” a malignant disease. The tumor antigen we have selected as a target is the HSP glucose-regulated protein of 94 kDa (Grp94), a member of the HSP90 family, since it plays an important role in the biology of malignant cells. Furthermore, we have recently developed a human monoclonal antibody (mAb), named mAb W9, which has a unique specificity. It recognizes an extracellular epitope of Grp94, which is expressed on many types of malignant cells, including differentiated pancreatic adenocarcinoma cells and pancreatic CICs. mAb W9 inhibited in vitro growth of the human pancreatic cancer cell lines PANC2.03 and PANC3.27 by ∼40%. The antiproliferative activity was increased to 90% when mAb W9 was combined with cyclopamine and 5-FU, both of which inhibit the hedgehog signaling pathway that is aberrantly activated in pancreatic adenocarcinoma, particularly in pancreatic CIC. The antitumor activity of mAb W9 reflected a decreased level of RAS, C-Raf, phosphorylated (p)-MEK, p-ERK, and p-AKT. mAb W9 had also an effect on pancreatic CICs, identified as cells with high aldehyde dehydrogenase expression. These cells are referred to as ALDHbright cells. Treatment of the human pancreatic cancer cell line MIA PaCa-2 in vitro with mAb W9, cyclopamine and 5-FU inhibited the growth of ALDHbright cells by ∼90%. In contrast, the inhibition was only 50% when cells were treated with mAb W9 or cyclopamine individually, 20% when they were treated with 5-FU and 70% when they were treated with mAb W9 in combination with cyclopamine or 5-FU. Apoptosis was induced in 70% of pancreatic cancer cells PANC1 by mAb W9 in combination with cyclopamine and 5-FU, but in less than 35% by the agents individually and by the combinations of any two of the agents. Our in vitro findings strongly suggest that targeting cell surface Grp94 with mAb W9 in combination with cyclopamine and 5-FU may represent an effective strategy to eliminate differentiated pancreatic cancer cells and pancreatic CICs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4390. doi:1538-7445.AM2012-4390

Mechanisms of Action of Human Aldehyde Dehydrogenase Bright Cells in Therapy of Cardiovascular Diseases: Expression Analysis of Angiogenic Factors and Aldehyde Dehydrogenase Isozymes

Human stem cell populations that express high aldehyde dehydrogenase activity [ALDHbr cells] have angiogenic activity in preclinical models and have been used safely to treat patients in early clinical trials. Bone marrow ALDHbr cells are being developed for therapeutic use in ischemic cardiovascular diseases. The mechanisms by which ALDHbr cells repair ischemic tissue are unknown, but available data suggest that angiogenic factors released by ALDHbr cells are involved. Gene expression studies were performed, and bone marrow ALDHbr cells were found to express 69 of 84 angiogenic factors tested. The 25 most highly expressed genes included soluble cytokines and growth factors, cytokine receptors, extracellular matrix proteins, and cell-cell signaling receptors. ALDHdim bone marrow cells that do not express high levels of ALDH and that have no angiogenic activity in preclinical models expressed a different group of genes. CD105 and Ephrin B4 transcripts were expressed about 65-fold more highly in ALDHbr than ALDHdim cells, and expression of these proteins was demonstrated in ALDHbr cells by flow cytometry. Expression analysis probing all 19 ALDH isozymes and immunofluorescence both demonstrated that ALDH1A1, an enzyme that can generate retinoic acids from retinaldehyde, was the ALDH isozyme most highly over expressed in ALDHbr cells compared to ALDHdim cells. Transwell studies demonstrated that ALDHbr cells responded to hypoxic conditions and to factors released from human endothelial vein cells (HUVEC) with changes in expression of specific angiogenic factors. Soluble factors released from ALDHbr cells in transwell cultures under hypoxic conditions stabilized endothelial tubules formed by HUVEC. The results are consistent with the hypothesis that human bone marrow ALDHbr cells can promote angiogenesis in ischemic tissues of patients with cardiovascular disease by several mechanisms including the release of soluble mediators.

SASP mediates chemoresistance and tumor-initiating-activity of mesothelioma cells

Here we show that pemetrexed-treated mesothelioma cells undergo accelerated senescence. This is characterized by the secretion of proinflammatory and mitogenic cytokines, reminiscent of an SASP (senescence-associated secretory phenotype). Conditioned media from senescent MPM (malignant pleural mesothelioma) cells trigger the emergence of EMT (epithelial-to-mesenchymal)-like, clonogenic and chemoresistant cell subpopulations, expressing high levels of ALDH (aldehyde dehydrogenase) activity (ALDH(bright) cells). We show by fluorescence-activated cell sorting of purified ALDH(bright) and ALDH(low) cells, that both cell-autonomous and cell-non-autonomous mechanisms converge to maintain the SASP-induced, EMT-like cell subpopulations. Chemoresistant ALDH(bright) cells exist within primary MPM specimens and enrichment for ALDH(bright) cells correlates with an earlier tumor onset into NOD/SCID mice. We show that RAS(v12) expression induces SASP-like changes in untransformed human mesothelial cells, and that p53 ablation increases the effect of RAS(v12) expression. We identify STAT3 activation as a crucial event downstream to SASP signaling. In fact, small hairpin RNA-mediated ablation of STAT3 deeply attenuates the induction of EMT genes and the increase of ALDH(bright) cells induced by SASP-cytokines. This strongly affects the chemoresistance of MPM cells in vitro and leads to anticancer effects in vivo.

Heterogeneity of leukemia stem cell candidates at diagnosis of acute myeloid leukemia and their clinical significance

Leukemia stem cell candidates (LSCC) can be enriched from patients with acute myeloid leukemia by high aldehyde dehydrogenase (ALDH) activity and CD34 expression. We have previously demonstrated the leukemia-initiating activity of ALDH(bright) cells in xenograft transplantation models, as well as invitro. Applying single-cell long-term culture-initiating cell assays, we have correlated the functional properties of individual cells within this LSCC population and the respective phenotypes. To define their biologic significance, we also analyzed the relationship between LSCC at diagnosis to long-term clinical outcomes. The median percentage of ALDH(bright) cells among 101 acute myeloid leukemia patients was 0.51% (range, 0.01-12.90%). Single-cell long-term culture-initiating cell assays, followed by genetic analysis of the progeny cells, showed that the leukemia-initiating activity was found in the ALDH(bright)/CD34(high) subset and, to a lesser extent, in ALDH(bright)/CD34(low) or ALDH(bright)/CD34(-) subsets. Nevertheless, the frequency of ALDH(bright) cells at diagnosis correlated significantly with the persistence of leukemia after induction chemotherapy (n = 84, Spearman R = 0.3261; p < 0.0025). In the multivariate model, frequency of ALDH(bright) cells was the strongest prognostic marker (p = 0.0095) affecting overall survival (hazard ratio = 9.107). LSCC are heterogeneous and best reflected by ALDH activity. The frequency of ALDH(bright) cells at diagnosis is a significant prognostic marker for acute myeloid leukemia.

Targeting ALDHbright Human Carcinoma–Initiating Cells with ALDH1A1-Specific CD8+ T Cells

Cancer-initiating cells (CIC) are considered to represent the subpopulation of tumor cells that is resistant to conventional cancer treatments, highly tumorigenic in immunodeficient mice, and responsible for tumor recurrence and metastasis. Based on an elevated aldehyde dehydrogenase (ALDH) activity attributable to ALDH1/3 isoforms, ALDH(bright) cells have been identified and isolated from tumors and shown to have characteristics of CIC. The ALDH1A1 isoform was previously identified as a tumor antigen recognized by CD8(+) T cells. This study examines the ability of ALDH1A1-specific CD8(+) T cells to eliminate ALDH(bright) cells and control tumor growth and metastases. ALDH(bright) cells were isolated by flow cytometry using ALDEFLUOR from HLA-A2(+) human head and neck, breast, and pancreas carcinoma cell lines and tested for their tumorigenicity in immunodeficient mice. ALDH1A1-specific CD8(+) T cells were generated in vitro and tested for their ability to eliminate CICs in vitro and in vivo by adoptive transfer to immunodeficient mice bearing human tumor xenografts. ALDH(bright) cells isolated by flow cytometry from HLA-A2(+) breast, head and neck, and pancreas carcinoma cell lines at low numbers (500 cells) were tumorigenic in immunodeficient mice. ALDH(bright) cells present in these cell lines, xenografts, or surgically removed lesions were recognized by ALDH1A1-specific CD8(+) T cells in vitro. Adoptive therapy with ALDH1A1-specific CD8(+) T cells eliminated ALDH(bright) cells, inhibited tumor growth and metastases, or prolonged survival of xenograft-bearing immunodeficient mice. The results of this translational study strongly support the potential of ALDH1A1-based immunotherapy to selectively target CICs in human cancer.

Highlights of This Issue

Administration of recombinant human erythropoietin (rHuEPO) has largely replaced bloodtransfusion in patients with cancer-induced anemia. Potential detrimental effects of thisapproach, such as thromboembolic risks and potential tumor response leading to enhanced disease progression, have become points of discussion and concern. Hedley and colleagues present data demonstrating potentiating effects on metastasis and reduction of chemotherapeutic efficacy in secondary sites by rHuEPO. This study begins to uncover the underlying functional explanation for the adverse events and decreased survival that have been observed in erythropoietin-treated metastatic breast cancer patients undergoing chemotherapy.Cancer-initiating cells (CIC) are being readily identified by elevated levels of aldehyde dehydrogenase (ALDH), namely ALDH1A1, a tumorassociated antigen recognized by HLA class I—restricted CD8+ T cells. Pertinent to developing immunotherapy for targeting CICs, Visus and colleagues demonstrate that ALDHbright cells are sensitive to cytolysis by ALDH1A1-specific CTLs in vitro and in vivo. In preclinical models of human tumor xenografts growing in immunodeficient mice, adoptive therapy with ALDH1A1-specific CD8+ T cells targeted ALDHbright cells and inhibited xenograft growth and metastasis or prolonged survival. These results highlight ALDH1A1 as a target of T cell—based immunotherapy to eliminate CICs.Androgen withdrawal therapy (AWT) is considered the standard of care for patients withmetastatic prostate cancer, but resistance to this therapy develops within 2 to 3 years after treatment. Chen and colleagues show that during AWT, increased levels of the receptor tyrosine kinases HER2 and ErbB3, members of the epidermal growth factor receptor (EGFR)family, result in a significant ErbB-dependent survival advantage that allows progressionto castrationresistant prostate cancer. However, dual EGFR/HER2 inhibition, which alsoinhibits their dimerization partner, ErbB3, induced apoptosis in cells undergoing AWT. Theseresults indicate that administration of dual EGFR/HER2 inhibitors together with AWT may impede the onset of castration-resistant prostate cancer.MicroRNAs (miRNAs) have critical functions across various biologic processes and can be potentially used as cancer biomarkers. Colorectal cancer is a heterogeneous disease, and the identification of various types of colorectal cancer with histopathologic methods alone is challenging. In this study, Balaguer and colleagues used miRNA expression profiling for molecular classification of hereditary and sporadic forms of colorectal cancer. Their data show that miRNA expression profiling not only distinguished between tumor and normal tissues, but more importantly, a subset of miRNAs successfully discriminated among Lynch syndrome, sporadic microsatelliteunstable, and microsatellite-stable colorectal cancers. These findings could have tremendous clinical and translational relevance in the diagnosis, prognosis, and treatment strategy for patients with different subtypes of colorectal cancer.

CD34 + Autologous Human Stem Cells in Treating Refractory Angina

See related article, pages 428–436 The article by Losordo et al1 in this issue of Circulation Research is an important addition to the relatively small body of literature in clinical cardiac stem cell therapy. Although the number of patients in the trial by Losordo et al is fairly small, only a handful of similarly sized trials have been published.2–4 In the recent past, the merit of performing clinical trials of cardiac cell therapy was debated in the literature.5,6 However, the study of regenerative cardiac medicine continues to move forward, despite the fact that opponents have highlighted the many challenges in the field, focusing mainly on the lack of mechanistic data. Nevertheless, other questions, such as the best cell type, cell dose, and delivery modality, are beginning to be answered. The performance of larger-scale studies is now being proposed. Interestingly, a large phase III trial of autologous bone marrow mononuclear cells (ABMMNCs) is ready to be initiated in Europe (Oral presentation by Anthony Mathur, PhD; June 2011, Eighth International Symposium on Stem Cell Therapy and Cardiovascular Innovations, Madrid, Spain). Most clinical trials in cardiac cell therapy have been performed with ABMMNCs. Although the use of this mixed cell population may be considered a relatively broad or less sophisticated approach, ABMMNCs continue to be used because of the previous success with this approach. However, investigators are also seeking new, possibly more potent cell types in bone marrow subpopulations, such as aldehyde dehydrogenase bright (ALDHbr) cells7 (characterized by a common enzyme), mesenchymal cells (a single bone marrow cell population), or as in the trial by Losordo et al, CD34+ …

Concise Review: Aldehyde Dehydrogenase Bright Stem and Progenitor Cell Populations from Normal Tissues: Characteristics, Activities, and Emerging Uses in Regenerative Medicine

Flow cytometry has been used to detect cells that express high levels of the aldehyde dehydrogenase activity in normal tissues. Such ALDH bright (ALDHbr) cell populations have been sorted from human cord blood, bone marrow, mobilized peripheral blood, skeletal muscle, and breast tissue and from the rodent brain, pancreas, and prostate. A variety of hematopoietic, endothelial, and mutiltipotential mesenchymal progenitors are enriched in the human bone marrow, cord, and peripheral blood ALDHbr populations. Multipotential neural progenitors are enriched in rodent brain tissue, and tissue-specific progenitors in the other tissue types. In xenograft models, uncultured human bone marrow and cord ALDHbr cells home to damaged tissue and protect mice against acute ischemic injury by promoting angiogenesis. Uncultured cord ALDHbr cells also deploy to nonhematopoietic tissues and protect animals in CCl4 intoxication and chronic multiorgan failure models. Mouse ALDHbr cells and cells derived from them in culture protect animals in a chronic neurodegenerative disease model. Purifying ALDHbr cells appears to increase their ability to repair tissues in these animal models. Clinical studies suggest that the number of ALDHbr cells present in hematopoietic grafts or circulating in the blood of cardiovascular disease patients is related to clinical outcomes or disease severity. ALDHbr cells have been used to supplement unrelated cord blood transplant and to treat patients with ischemic heart failure and critical limb ischemia. ALDH activity can play several physiological roles in stem and progenitor cells that may potentiate their utility in cell therapy.

Mammosphere-forming cells from breast cancer cell lines as a tool for the identification of CSC-like- and early progenitor-targeting drugs

Here we show that a subpopulation of MCF-7 breast cancer cells which stains pale to Toluidine Blue (Light Cells- LCs), is endowed with features of CSCs. LCs give rise to self-renewing mammospheres and express typical CSC markers; moreover this subpopulation is chemoresistant and highly tumorigenic in vivo. LCs can be identified in several other breast cancer cell lines, irrespectively of their histological origin (luminal vs mesenchymal vs basal) and represent an heterogeneous cell population composed mainly of CSC-like and early progenitor cells. By a limited in vitro drug screening assay, we identify compounds which can specifically interfere with the viability of LCs from multiple breast cancer cell lines. Analysis of the Sphere-Forming Efficiency (SFE) and of the distribution of ALDHbright cells within the treated cell lines suggest that one of the identified compounds acts in vitro by modulating the CSC phenotype. Interestingly, a subset of the identified compounds is known to affect directly or indirectly the NFkB pathway which is emerging as an important modulator of CSC proliferation and chemoresistance.

Abstract 3339: Elimination of cancer stem cells in pancreatic cancer cell lines by a combinatorial therapy

Abstract Cancer stem cells (CSC) are tumor initiating cells and considered resistant to current conventional chemo and radio therapies. Consequently, novel strategies are needed to target these cells. Tumor cells expressing elevated levels of aldehyde dehydrogenase 1 family member A1 activity (ALDHbright cells) have been shown to have many properties attributed to CSC, including tumorigenicity in immunodeficient mice. We used FACS analysis to identify and sort ALDHbright cells from several human pancreatic cell lines using ALDEFLUOR (Stem Cell Technologies, Vancouver Canada). We show that ALDHbright but not ALDHnegative cells sorted from the human MiaPaCa-2 pancreatic adenocarcinoma cell are highly tumorigenic at low inoculum of 500 cells in immunodeficient NOD.SCID mice, a critical property of CSC. Previously, we identified the ALDH1A188–96 peptide as an HLA-A2-restricted, cytotoxic T cell (CTL)-defined tumor antigen. Using a flow cytometry-based assay, ALDHbright cells in the HLA-A2+ MiaPaCa-2 cell line were selectively eliminated in vitro by ALDH1A188–96 peptide-specific CTL. The Hedgehog (Hh) stem cell signaling pathway has been shown to play a critical role in invasion and metastasis in pancreatic cancer. Treatment of the MiaPaCa-2 cell line with the Hg inhibitor, cyclopamine, significantly eliminated ALDH+ MiaPaCA-2 cells compared to treatment with conventional chemotherapeutic agents. The efficacy of a combinatorial therapy consisting of adoptive transfer of ALDH1A1-specific CTL combined with systemic administration of cyclopamine in vitro and in vivo on ALDHbright cells and in vivo growth inhibition of MIA PaCa-2-derived xenografts is detailed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3339.

Abstract 5605: Targeting cancer stem cells in squamous cell carcinoma of the head and neck with combinatorial adoptive immunotherapy

Abstract According to current views, in order for cancer immunotherapy to be successful it must target cancer stem cells (CSC) and counteract the multiple mechanisms involved in immune escape. Guided by these concepts, we have developed a combinatorial adoptive immunotherapeutic strategy which utilizes a monoclonal antibody (mAb) and cytotoxic T lymphocytes (CTL) in combination with continuous administration of low dose cyclophosphamide (metronomic chemotherapy) to target CSC and differentiated tumor cells, as well as activated pericytes in the tumor microenvironment. The targets selected are aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and chondroitin sulfate proteoglycan 4 (CSPG4). We now report that ALDHbright cells in squamous cell carcinoma of the head and neck (SCCHN) cell lines, which express high levels of ALDH1A1, are tumorigenic at low inoculum in immunodeficient mice, and particularly sensitive to recognition by ALDH1A1-specific T cells. The ALDHbright cells express CSPG4, a cell surface antigen functional in adhesion and migration. CSPG4 is also expressed on tumor associated activated pericytes which stabilize tumor blood vessels and provide vascular endothelial cells with survival signals. Administration of CSPG4-specific mAb and metronomic chemotherapy combined with intralesional adoptive transfer of ALDH1A1 peptide-specific CTL to mice bearing human SCCHN PCI-13-derived xenografts significantly reduced tumor growth and number of tumor cells with CSC phenotype. The antitumor effects of the immunotherapy we have developed were significantly higher than those of its individual components. These results argue in favor of the possibility that this combinatorial strategy may enhance the clinical efficacy of adoptive immunotherapy in SCCHN patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5605.