AC133 Epitope Research Articles

Enhanced AC133-specific CAR T cell therapy induces durable remissions in mice with metastatic small cell lung cancer

Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133+ cancer stem cells and PD-L1+CD73+ myeloid cells were detectable in primary human SCLC tissues, suggesting that patients may benefit from the triple-immunotherapy. We conclude that the combination of AC133-specific CAR T cells, anti-PD-1-antibody and CD73-inhibitor specifically eliminates chemo-resistant tumor stem cells, overcomes SCLC-mediated T cell inhibition, and might induce long-term complete remission in an otherwise incurable disease.

A possible interplay between HR-HPV and stemness in tumor development: an in vivo investigation of CD133 as a putative marker of cancer stem cell in HPV18-infected KB cell line.

High-risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence, and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. We started our study by identifying the most aggressive cell line on the minimal number of cells being able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analyzed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133-enriched xenograft tumors versus the CD133 non-enriched ones. On standard conditions, the KB cell line has a poor population of glycosylated CD133 marker (<5.0%) when investigated with antibodies versus CD133, and more specifically its glycosylated epitope (AC133). Enriched CD133 KB cells possess a higher capacity of tumor growth in xenograft models of nude mice when compared to KB CD133-negative cells. We observed that the AC133 epitope, extensively used to purifying hematopoietic stem cells, is able to select an epithelial subpopulation of cancer stem cells with aggressive behavior. We retain that CD133 may be a useful target in anticancer strategies including pharmacological and immunological therapies.

CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells.

A transmembrane protein CD133 has been implicated as a marker of stem-like glioma cells and predictor for therapeutic response in malignant brain tumours. CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for identifying stem-like glioma cells and predicting the degree of malignancy in glioma cells. The reasons for discrepant results between different studies addressing the role of CD133/AC133 in gliomas are unclear. A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein. Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein. The purpose of this study is to determine whether and to what extent do the readouts obtained with anti-AC133 antibody correspond to the level of CD133 protein expressed in stem-like glioma cells. Our study reveals for the first time that CD133 expressed on the surface of glioma cells is poorly immunoreactive for AC133. Furthermore, we provide evidence that the level of CD133 occupancy on the surface of glioma cells fluctuates during the cell cycle. Our results offer a new explanation for numerous inconsistencies regarding the biological and clinical significance of CD133/AC133 in human gliomas and call for caution in interpreting the lack or presence of AC133 epitope in glioma cells.

Effective Eradication of Glioblastoma Stem Cells by Local Application of an AC133/CD133-Specific T-cell-Engaging Antibody and CD8 T Cells.

Cancer stem cells (CSC) drive tumorigenesis and contribute to genotoxic therapy resistance, diffuse infiltrative invasion, and immunosuppression, which are key factors for the incurability of glioblastoma multiforme (GBM). The AC133 epitope of CD133 is an important CSC marker for GBM and other tumor entities. Here, we report the development and preclinical evaluation of a recombinant AC133×CD3 bispecific antibody (bsAb) that redirects human polyclonal T cells to AC133(+) GBM stem cells (GBM-SC), inducing their strong targeted lysis. This novel bsAb prevented the outgrowth of AC133-positive subcutaneous GBM xenografts. Moreover, upon intracerebral infusion along with the local application of human CD8(+) T cells, it exhibited potent activity in prophylactic and treatment models of orthotopic GBM-SC-derived invasive brain tumors. In contrast, normal hematopoietic stem cells, some of which are AC133-positive, were virtually unaffected at bsAb concentrations effective against GBM-SCs and retained their colony-forming abilities. In conclusion, our data demonstrate the high activity of this new bsAb against patient-derived AC133-positive GBM-SCs in models of local therapy of highly invasive GBM.

Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57.

The AC133 epitope of CD133 is a cancer stem cell (CSC) marker for many tumor entities, including the highly malignant glioblastoma multiforme (GBM). We have developed an AC133-specific chimeric antigen receptor (CAR) and show that AC133-CAR T cells kill AC133+ GBM stem cells (GBM-SCs) both in vitro and in an orthotopic tumor model in vivo. Direct contact with patient-derived GBM-SCs caused rapid upregulation of CD57 on the CAR T cells, a molecule known to mark terminally or near-terminally differentiated T cells. However, other changes associated with terminal T cell differentiation could not be readily detected. CD57 is also expressed on tumor cells of neural crest origin and has been preferentially found on highly aggressive, undifferentiated, multipotent CSC-like cells. We found that CD57 was upregulated on activated T cells only upon contact with CD57+ patient-derived GBM-SCs, but not with conventional CD57-negative glioma lines. However, CD57 was not downregulated on the GBM-SCs upon their differentiation, indicating that this molecule is not a bona fide CSC marker for GBM. Differentiated GBM cells still induced CD57 on CAR T cells and other activated T cells. Therefore, CD57 can apparently be upregulated on activated human T cells by mere contact with CD57+ target cells.

Post-Translational Regulation of CD133 by ATase1/ATase2-Mediated Lysine Acetylation

The CD133 cell-surface protein expresses the AC133 epitope that is associated with cancer progenitor cells and cancer resistance to traditional anticancer therapies. We report that the endoplasmic reticulum Golgi intermediate compartment residing acetyltransferases, ATase1 (NAT8B) and ATase2 (NAT8), can physically interact with CD133 to acetylate the protein on three lysine residues predicted to reside on the first extracellular loop of CD133. Site-directed mutagenesis of these residues mimicking a loss of acetylation and downregulation or inhibition of ATase1/ATase2 resulted in near-complete abolishment of CD133 protein expression. We also demonstrate that targeting ATase1/ATase2 results in apoptosis of CD133 expressing acute lymphoblastic leukemia cells. Taken together, we suggest that lysine acetylation on predicted extracellular residues plays a key role in expression and trafficking of CD133 protein to the cell surface and can be targeted to disrupt CD133 regulation and function.

Noninvasive positron emission tomography and fluorescence imaging of CD133 + tumor stem cells

A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133(+) tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with (64)Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2-3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133(+) cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133(+) tumor stem cells.

CD133 glycosylation is enhanced by hypoxia in cultured glioma stem cells

The cancer stem cell (CSC) marker CD133 is widely expressed in gliomas and employed mostly by use of the CD133/1 antibody which binds the extracellular glycosylated AC133 epitope. CD133 recognition may, however, be affected by its glycosylation pattern and oxygen tension. The present study investigates the effect of oxygen deprivation on CD133 expression and glycosylation status employing a high AC133-expressing glioblastoma multiforme (GBM) cell line, IN699. IN699 cells were cultured under normoxic (21% O2) and hypoxic (3% O2) conditions. CD133 expression was analysed by western blotting (WB), qRT-PCR, immunocytochemistry (ICC) and flow cytometry using the glycosylation-specific antibody CD133/1 and ab19898 which binds the unglycosylated intra-cellular residues of CD133. By flow cytometry, ab19898 detected 94.1% and 96.2% CD133+ cells under normoxia and hypoxia, respectively. Hypoxia significantly increased the percentage of CD133+ cells from 69% to 92% using CD133/1 (p<0.005). Moreover, a significantly higher geomean fluorescence intensity (GMI) was demonstrated by ab19898 (p<0.005) in CD133+ cells. WB and qRT-PCR results were consistent with flow cytometry data. Furthermore, over a period of 72-h incubation under normoxic and hypoxic conditions after autoMACS sorting, an average of 31.8% and 42.2%, respectively, of CD133-negative IN699 cells became positive using CD133/1. Our data show that a) previously reported CD133- cells may have been misidentified using the glycosylation-specific CD133/1 as constitutive expression of CD133 was detected by the intracellular antibody ab19898; b) hypoxia promotes glycosylation status of CD133, indicating possible involvement of glycosylated CD133 in the process of anti-hypoxia-mediated apoptosis.

AC133 expression associated with poor prognosis in stage II colorectal cancer

Identification of high-risk prognostic markers for stage II colorectal cancer (CRC) is currently a big challenge. CD133 is one of the most commonly used CRC stem cell markers. However, its specificity is controversial. Recent studies have demonstrated that the AC133 epitope of CD133, not the CD133 protein, is responsible for cancer stem cell identification. The aim of this study was to investigate the clinical significance of AC133 expression in stage II CRC. Two antibodies against CD133, including AC133 and Ab19898, were compared for their expression characteristics. AC133 was chosen for further immunohistochemical assessment on 176 stage II CRC primary tumors with at least 12 examined lymph nodes. The cutoff value for positive rate of AC133 expression was determined by ROC curve analysis. AC133 was analyzed for correlations with clinicopathological and prognostic parameters. The results indicated that AC133 was negative in adjacent noncancerous colorectal mucosa while positive in 116 cases (65.9 %) of primary tumors. AC133 expression was significantly correlated with preoperative serum carcinoembryonic antigen level (p = 0.006) and tumor differentiation grade (p = 0.019). Furthermore, high AC133 expression was identified as a significant predictor for poor disease-free survival and overall survival at both univariate (p = 0.009, 0.013, respectively) and multivariate levels (p = 0.022, 0.026, respectively). Our data suggest that AC133 is an independent adverse prognostic factor and a potential marker for survival classification in stage II CRC patients.

RNA aptamers targeting cancer stem cell marker CD133

The monoclonal antibody against the AC133 epitope of CD133 has been widely used as a cell surface marker of cancer stem cells in several different cancer types. Here, we describe the isolation and characterisation of two RNA aptamers, including the smallest described 15 nucleotide RNA aptamer, which specifically recognise the AC133 epitope and the CD133 protein with high sensitivity. As well, both these aptamers show superior tumour penetration and retention when compared to the AC133 antibody in a 3-D tumour sphere model. These novel CD133 aptamers will aid future development of cancer stem cell targeted therapeutics and molecular imaging.

CD133 as a biomarker for putative cancer stem cells in solid tumours: limitations, problems and challenges

The cancer stem cell (CSC) hypothesis, despite the limitations of the currently available models and assays, has ushered in a new era of excitement in cancer research. The development of novel strategies for anti-tumour therapy relies on the use of biomarkers to identify, enrich, and/or isolate the cell population(s) of interest. In this context, various cell characteristics and antigen expression profiles are discussed as surrogate markers. The cell surface expression of the human prominin-1 (CD133) antigen, in particular of the AC133 epitope, is among those that have been most frequently studied in solid cancers, although no mechanism has yet been proposed to link CD133 expression with the CSC phenotype. Some inconsistencies between published data can be ascribed to different analytical tools as well as methodological limitations and pitfalls, highlighted in the present review. Therefore, a comprehensive overview on the current state of knowledge in this growing and exciting field with an emphasis on the most recent studies is presented. We highlight the link between the tumour microenvironment, tumour cell plasticity, and CD133 expression, and evaluate the utility of CD133 expression as a prognostic marker.

Expression of CD133 mRNA splice variants in glioma

Objective To determine the relationship between CD133 mRNA splice variant and AC133 epitope.Methods The expression of CD133 mRNA splice variants was compared between AC133 positive and negative glioma cells under different culture conditions or by FACS sort.The splice variants expression in AC133 positive glioms stem cells under differentiation was also studied. RT - PCR was performed to specifically amplify the CD133 mRNA sequence containing facultative exons,which were further sequenced to determine the expressed facultative exons. Meanwhile,the real - time quantitative fluorescent PCR was performed to quantify the facultative exons.Results AC133 positive and negative glioma cells from different culture condition or by FACS sort expressed the same splice variants. No correlation between splice variants expression and CD133 protein sub - cellular location was found.The relative expression of splice variants containing facultative exon3 was up - regulated in AC133 positive glioma stem cells under differentiation( P ＜ 0.05 ).Conclusions In glioma no specific CD133 mRNA splice variant for AC133 epitope is identified. Splice variants containing facultative exon3 correlate with the differentiated phenotype of glioma cells. Key words: Glioma; Neoplastic stem cell; Antigens, CD133; Splice variant

Abstract 5340: Standardization of cancer stem cell enumeration and isolation methods allows cross comparison among different tumor entities

Abstract Growing knowledge concerning tumor heterogeneity has increased the importance of analyzing tumor resident cell and stem cell subpopulations. In particular, cancer stem cells (CSCs), also called tumor initiating cells, have gained substantial interest in the research field over the past few years. CSCs have been isolated from multiple tumor entities and were shown to play a crucial role during tumor growth and metastasis. However, isolation and analysis of CSCs even from single tumor entities like melanoma showed contradictory results among different research groups. This can at least partially be explained by variations in the dissociation and isolation methods used. One critical point that has to be considered during the analysis of putative CSC markers is that the procedure used for dissociation of primary tissue prior to staining may cause a technical bias. Aggressive proteases, such as trypsin, which cleave off several CSC surface markers like ABCB5 or CD44, may cause incorrect results concerning the differential expression of sensitive markers (Civenni et al., 2011). A further source of non-reproducible findings is the use of antibodies that are specific to sub-forms of cell surface markers. Glycosylation and splice-isoform dependent epitopes, as found for e.g. CD24 and CD133, are differentially expressed among tissues and cell states. In particular, it was shown that the AC133 epitope but not the entire CD133 protein expression is lost upon CSC differentiation (Kemper et al., 2010). We have developed a standardized platform that allows fast and highly reproducible CSC enumeration and isolation from various tumor tissues. This platform includes automated and therefore user independent procedures for dissociation of human and implanted mouse tumor tissue without degradation of cell surface epitopes yielding high amounts of single cells at viabilities around 90%. Furthermore, antibody conjugates of clones recognizing epitopes shown to be relevant for CSC analysis were generated and tested on cell lines as well as primary tumor tissue allowing for the quantification and isolation of CSCs. As a proof of principle, highly viable populations of CD133+ and CD45−/CD24−/CD44+ CSCs were efficiently isolated from a glioblastoma at 86% and an invasive ductal mamma carcinoma at 94% purity, respectively. In addition, optimized gating and exclusion strategies are presented avoiding analytical bias for flow cytometry-based CSC quantification from primary tissue as shown for human melanoma tissue. By using reliable methods for the analysis of CSCs from various origins, a major cause of conflicting results in this field may be minimized and comparisons of these subpopulations among distinct tumor entities will be possible. 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 5340. doi:1538-7445.AM2012-5340

The Mixed Lineage Leukemia (MLL) Fusion–Associated Gene AF4 Promotes CD133 Transcription

The AC133 epitope has been used as a marker for both normal and cancer stem cells from multiple tissue lineages. To identify transcription factors that regulate CD133 expression, we conducted parallel large-scale RNA interference screens in Caco-2 cancer cells that endogenously express CD133 and in engineered HEK293 cells that express CD133 from a heterologous promoter. The transcription factor AF4 was identified following a comparative analysis between the two screens. We then showed that AF4 is a promoter of CD133 transcription in multiple cancer cell lines. Knockdown of AF4 resulted in a dramatic reduction in CD133 transcript levels. Importantly, a subset of pediatric acute lymphoblastic leukemias (ALL) harbor a fusion oncogene results from a chromosomal translocation that juxtaposes the mixed-lineage leukemia (MLL) gene and the AF4 gene. An investigation of the functional role of CD133 in the MLL-AF4-dependent ALL cells revealed that CD133 was required for leukemia cell survival. Together, our findings show AF4-dependent regulation of CD133 expression, which is required for the growth of ALL cells. CD133 may therefore represent a therapeutic target in a subset of cancers.

CD133 Protein N-Glycosylation Processing Contributes to Cell Surface Recognition of the Primitive Cell Marker AC133 Epitope

The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.

Expression and regulation of AC133 and CD133 in glioblastoma

The biological significance of CD133 in glioblastoma is controversial. Above all, there is disagreement concerning the proper approach, the appropriate (cell) model and the suitable microenvironment to study this molecule, often leading to inconsistent experimental results among studies. In consideration of a primary need to dissect and to understand the CD133 phenotype in glioblastoma we performed a comprehensive analysis of CD133 expression and regulation in a large set of glioblastoma cell lines (n = 20) as well as in tumor xenografts. Our analysis considered alternatively spliced mRNA transcripts, different protein epitopes as well as varying sub-cellular localizations of CD133 and explored its regulation under pertinent micro-environmental conditions. CD133 mRNA and CD133 protein could be detected in all relevant types of glioblastoma cell lines. In addition, we detected frequent intracellular CD133 protein accumulations located to the ER and/or Golgi apparatus but seemingly unrelated to particular CD133 splice variants or protein epitopes. In contrast, membrane-bound expression of CD133 was restricted to tumor cells bearing the extracellular CD133 epitope AC133. Only in these cells, differentiation and oxygen levels clearly impacted on AC133 expression and to some extent also influenced CD133 mRNA and protein expression. Most importantly, however, modulation of AC133 levels could occur independently of changes in CD133 mRNA transcription, CD133 protein translation, protein retention or protein shedding. Our results suggest that the AC133 epitope, rather than CD133 mRNA or protein, mirrors malignancy-related tumor traits such as tumor differentiation and local oxygen tension levels, and thus corroborate its role as a biologically relevant cancer marker.

Prominin‐1 (CD133) is not restricted to stem cells located in the basal compartment of murine and human prostate

Rodent and human prominin-1 are expressed in numerous adult epithelia and somatic stem cells. A report has shown that human PROMININ-1 carrying the AC133 epitope can be used to identify rare prostate basal stem cells (Richardson et al., J Cell Sci 2004; 117:3539–3545). Here we re-investigated its general expression in male reproductive tract including mouse and human prostate and in prostate cancer samples using various anti-prominin-1 antibodies. The expression was monitored by immunohistochemistry and blotting. Murine tissues were stained with 13A4 monoclonal antibody (mAb) whereas human samples were examined either with the AC133 mAb recognizing the AC133 glycosylation-dependent epitope or 80B258 mAb directed against the PROMININ-1 polypeptide. Mouse prominin-1 was detected at the apical domain of epithelial cells of ductus deferens, seminal vesicles, ampullary glands, and all prostatic lobes. In human prostate, immunoreactivity for 80B258, but not AC133 was revealed at the apical side of some epithelial (luminal) cells, in addition to the minute population of AC133/80B258-positive cells found in basal compartment. Examination of prostate adenocarcinoma revealed the absence of 80B258 immunoreactivity in the tumor regions. However, it was found to be up-regulated in luminal cells in the vicinity of the cancer areas. Mouse prominin-1 is widely expressed in prostate whereas in human only some luminal cells express it, demonstrating nevertheless that its expression is not solely associated with basal stem cells. In pathological samples, our pilot evaluation shows that PROMININ-1 is down-regulated in the cancer tissues and up-regulated in inflammatory regions.

The AC133 Epitope, but not the CD133 Protein, Is Lost upon Cancer Stem Cell Differentiation

Colon cancer stem cells (CSC) can be identified with AC133, an antibody that detects an epitope on CD133. However, recent evidence suggests that expression of CD133 is not restricted to CSCs, but is also expressed on differentiated tumor cells. Intriguingly, we observed that detection of the AC133 epitope on the cell surface decreased upon differentiation of CSC in a manner that correlated with loss of clonogenicity. However, this event did not coincide with a change in CD133 promoter activity, mRNA, splice variant, protein expression, or even cell surface expression of CD133. In contrast, we noted that with CSC differentiation, a change occured in CD133 glycosylation. Thus, AC133 may detect a glycosylated epitope, or differential glycosylation may cause CD133 to be retained inside the cell. We found that AC133 could effectively detect CD133 glycosylation mutants or bacterially expressed unglycosylated CD133. Moreover, cell surface biotinylation experiments revealed that differentially glycosylated CD133 could be detected on the membrane of differentiated tumor cells. Taken together, our results argue that CD133 is a cell surface molecule that is expressed on both CSC and differentiated tumor cells, but is probably differentially folded as a result of differential glycosylation to mask specific epitopes. In summary, we conclude that AC133 can be used to detect cancer stem cells, but that results from the use of this antibody should be interpreted with caution.

Abstract A60: Targeting brain tumor stem cells

Abstract The brain tumor stem-cell (BTSC) hypothesis proposes with acquired self-renewal properties establish a brain tumorigenic-behavior (1). Thus, failure to cure brain cancer has been attributed to the fact that typical cytotoxic therapies target rapidly proliferating brain tumor cells, while sparing the BTSC compartment, which has a low proliferation rate and high tumorigenic potential. Taking advantage of the recent development of nanotechnologies that may offer innovative drug-delivery systems for improved efficacy, specificity and biological safety (2), the aim of our work is to develop nanoparticle-based treatments targeting BTSCs. The first part of our work represents a comprehensive study of the aggressiveness of the BTSC was shown to be associated with the expression of the neural precursor cell marker AC133. Hence, the percentage of AC133 expressing glioma cells is increased after maintenance or exposure to moderately low oxygen levels (3). Our data indicate that cells maintained at 3% oxygen express more AC133 and are more aggressive after in vivo xenograft than cells cultured at 21% oxygen. Thus, oxygen pressure represents a major parameter in the expression of the AC133 molecule but is also important in aggressiveness of glioma cells. The mechanism that links hypoxia, AC133 expression and the tumorigenic behavior of BTSC has been studied. Our results, including CoCl2 induction and shRNA knockdown, suggest a role for HIF-1α in the regulation of AC133 in glioblastoma cells. The second part of our work, immunonanoparticules (ILNC) that target AC133 were synthesized by conjugation of thiolated AC133 or IgG1 isotype control immunoglobulins to lipid nanocapsules (LNC) (4) by linkage through incorporation of lipid PEG(2000) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) on the surface of LNC by using a post-insertion procedure (5). Our results indicate a fixation yield of antibodies on LNC varying from 6 to 60% representing 70 antibodies per LNC. To evaluate the selectivity of immunonanoparticles binding and resulting uptake by cells a second in vitro model has been developed. Human Caco-2 cells, previously described to express AC133, were used and exposed to siRNA targeting the CD133 gene to dispose of two types of Caco-2 cells: Caco-2-AC133high and Caco-2-AC133low. We are now testing ILNC uptake in our Caco-2 cell model. We will test if ILNC are able to bind to AC133 epitope on Caco-2 cells and if they are able to be internalized in cells. Accumulated results must allow us to extend our knowledge on glioma aggressiveness and to adapt our vectors to resolve the BTSC targeting problem and their death. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A60.

Stem Cell Marker Prominin-1/AC133 Is Expressed in Duct Cells of the Adult Human Pancreas

Many efforts are spent in identifying stem cells in adult pancreas because these could provide a source of beta cells for cell-based therapy of type 1 diabetes. Prominin-1, particularly its specific glycosylation-dependent AC133 epitope, is expressed on stem/progenitor cells of various human tissues and can be used to isolate them. We, therefore, examined its expression in adult human pancreas. To detect prominin-1 protein, monoclonal antibody CD133/1 (AC133 clone), which recognizes the AC133 epitope, and the alphahE2 antiserum, which is directed against the human prominin-1 polypeptide, were used. Prominin-1 RNA expression was analyzed by real-time polymerase chain reaction. We report that all duct-lining cells of the pancreas express prominin-1. Most notably, the cells that react with the alphahE2 antiserum also react with the AC133 antibody. After isolation and culture of human exocrine cells, we found a relative increase in prominin-1 expression both at protein and RNA expression level, which can be explained by an enrichment of cells with ductal phenotype in these cultures. Our data show that pancreatic duct cells express prominin-1 and surprisingly reveal that its particular AC133 epitope is not an exclusive stem and progenitor cell marker.