CD133 Surface Expression Research Articles

Abstract 1154: Investigating the functional role of GPNMB in glioblastoma and the tumor immune microenvironment and its targeted elimination using CAR-Ts

Abstract Glycoprotein nonmetastatic melanoma protein B (GPNMB) is known to be active in the extracellular matrix of glioblastoma and has been identified as a promising immunotherapy target for both tumor cells and immunosuppressive macrophages. Methods: Immunohistochemistry was performed on patient derived xenograft (PDX) brains and tissue samples of 16 patient matched primary/recurrent GBMs as well as 23 normal organ tissues. Whole cell proteomics was performed on 43 matched primary/recurrent GBM samples. Flow cytometry measured surface expression levels of GPNMB to confirm CAR-T accessibility. CRISPR/Cas9 was used to eliminate expression in GBM lines to measure proliferation and mouse survival times. GPNMB knockout clones were generated in GL261 and engrafted in immunocompetent mice to examine single cell transcriptomes using sciRNAseq at endpoint. A second-generation CAR-T was developed to target GPNMB-expressing populations, and efficacy was interrogated using standard in vitro assays and GBM PDX models. Results: The absence of GPNMB throughout most normal tissues validates the rationale of administering CAR-Ts as a safe modality for patients. GPNMB detected in residual tumors of PDX models treated orthotopically with CD133 CAR-Ts revealed it as a targetable subpopulation of GBM cells and a rational co-target alongside CD133 in the heterogeneous tumor. Tissue microarrays and whole cell proteomics found GPNMB to be upregulated in recurrent GBMs compared to primary (p=0.0349 and p=0.0033 respectively) while being absent in normal tissues. Single cell sequencing data of patient GBMs revealed GPNMB was also highly expressed in tumor-associated macrophages. Eliminating GPNMB in GBM cell lines decreased proliferation (P<0.001) and prolonged survival times in all mouse models (P<0.01) indicating its functional relevance. GPNMB knockout clones displayed downregulation of hallmark signalling pathways of GBM such as PDGFR, TGF-beta, Integrins and Stats, as well as decreased innate/adaptive immune activation. CAR-T cytotoxicity and activation was observed in vitro and in vivo resulting in decreased tumor burden (P<0.001) and increased survival times (P<0.001). Ultimately a CD133+ population was observed in residual tumors of GPNMB CAR-T treated mice at endpoint and surface expression of CD133 and GPNMB revealed co-expression and distinct populations. Conclusions: We show GPNMB influences tumor-intrinsic biology of GBM and is also active in macrophages in the recurrent GBM immune microenvironment. By targeting GPNMB along with CD133, combinatorial therapeutic regimens could target both the cancer stem cell hierarchy and its supportive niche. Administration of both CAR-T cell therapies to humanized mice engrafted with patient-derived GBMs will provide better cytotoxic coverage and potentially provide more durable therapeutic efficacy for GBM patients. Citation Format: Neil Savage, Franz J. Zemp, Nick Mikolajewicz, Hong Han, Chitra Venugopal, Chirayu Chokshi, Nazanin Tatari, Thomas Kislinger, Jason Moffat, Doug Mahoney, Sheila K. Singh. Investigating the functional role of GPNMB in glioblastoma and the tumor immune microenvironment and its targeted elimination using CAR-Ts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1154.

Inhibition of Pim Kinase Supports the Neural Stem Cell Phenotype in Neuroblastoma

INTRODUCTION: Relapse rates for high-risk neuroblastoma exceed 50%. Expression of the serine-threonine protein kinase, PIM, is associated with cancer progression and is negatively correlated with neural stemness markers in neural progenitor cells. Because promoting cellular differentiation is a component of neuroblastoma therapy, and PIM kinase negatively affects neural stemness, we aimed to determine whether PIM affected the neural stem cell phenotype in neuroblastoma. METHODS: We queried the Kocak (R2) gene database to compare PIM1 and the neural stemness marker, Nanog, in neuroblastoma. For in vitro studies, SK-N-BE(2), human neuroblastoma cells and human neuroblastoma patient–derived xenograft (PDX06) cells were treated with siRNA to achieve PIM1 kinase knockdown or with AZD1208 for PIM inhibition. Abundance of mRNA for the neural stemness marker, Nanog, was detected with quantitative reverse transcription polymerase chain reaction, and protein expression was confirmed with immunoblotting. Flow cytometry detected cell surface expression of CD133 and CXCR4, 2 additional neural stemness markers. Data were reported as mean ± SEM, with p ≤ 0.05 considered significant. RESULTS: R2 database revealed a negative correlation between PIM1 and Nanog (Fig. 1A). Protein expression of Nanog was increased following siRNA knockdown of PIM1 (Fig. 1B). AZD1208 increased the abundance of Nanog mRNA and resultant protein expression (Fig. 1C). AZD1208 treatment significantly increased cell surface expression of CD133 and CXCR4 (Fig. 1D).Figure 1CONCLUSION: Neuroblastoma cells treated with PIM inhibition dedifferentiated to a more neuronal stem cell phenotype as demonstrated by increased expression of neuronal stemness markers. These findings support further studies exploring the clinical implications of PIM kinase inhibition as an adjunct to neuroblastoma therapy.

STEM-09. DEFINING THE ROLE OF CD97 IN GLIOBLASTOMA STEM CELL SELF-RENEWAL

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Despite multimodal therapy, resistant GBM stem-like cells (GSCs) inevitably mediate disease recurrence. To identify novel vulnerabilities of GSCs, we performed an arrayed CRISPR/Cas9 screen against select adhesion G protein-coupled receptors (aGPCRs), many of which we found to be de novo expressed in GBM. Knockout of CD97 (ADGRE5), previously implicated in GBM cell migration, produced the most striking proliferative disadvantage in patient-derived GBM cultures (PDGC) among aGPCRs tested. We found high CD97 surface expression in all our PDGCs, while levels remained nearly undetectable in non-neoplastic brain cells, confirming that CD97 is de novo expressed in GBM. Upon shRNA-mediated knockdown of CD97 in PDGCs from all three TCGA transcriptional subtypes, we observed significantly reduced proliferation, as measured by Ki67 and Hoechst cell cycle analysis, and significantly diminished surface expression of CD133, a GSC marker. Notably, CD97 knockdown also significantly reduced tumorsphere initiation capacity in six PDGCs, as measured by extreme limiting dilution assays. These findings suggest that CD97 regulates GSC self-renewal in vitro. RNA-sequencing and GSEA pathway analysis from PDGCs following CD97 knockdown indicate an enrichment of aerobic respiratory gene sets, suggesting one of the major regulatory roles of CD97 is metabolic regulation. Indeed, metabolic assays show that CD97 knockdown alters oxygen consumption and glycolysis rates in PDGCs. Lastly, we have developed human synthetic antibodies to target CD97 in order to investigate its therapeutic potential. We have observed internalization of some of these antibodies, thus identifying candidates for the development of antibody-drug conjugates. In addition, other clones reduced GBM cell proliferation and elicited expression of various differentiation markers. Overall; our studies identify novel roles of CD97 in regulating the cellular hierarchy in GBM and tumor cell metabolism, and provide a strong scientific rationale for developing biologics to target CD97 in GBM.

CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells.

Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.

The combination effect of Prominin1 (CD133) suppression and Oxaliplatin treatment in colorectal cancer therapy

Colorectal cancer (CRC) is considered one of the leading types of cancer in the world. CD133, as a cancer stem cell marker, has a pivotal role in the development of drug resistance, migration, and stemness properties of CRC cells. This study was designed to check the combined effect of CD133 siRNA and Oxaliplatin on proliferation, migration, apoptosis, and stemness properties of CRC cells in the HT-29 cell line. MTT assay was performed to define the combined effect of CD133 siRNA and Oxaliplatin on the viability of HT-29 cells, and it showed that the combination of CD133 siRNA and Oxaliplatin could reduce the IC50 of this drug from 32.85 to 19.75 nmol. In order to figure out the effect of this combination therapy on CD133 expression at the gene and protein level, qRT-PCR and western blot were exploited, respectively. The results demonstrated that the silencing of CD133 could reduce the relative expression of this marker to about 0.00001 compared to the control group and reduce the protein level to 0.01. The ability of cell migration was tested by wound healing assay as well. Also, colony formation and sphere formation were conducted to assess the stemness properties in the combination group. Flow cytometry was conducted to investigate the apoptosis (15%), cell cycle (about 10% arresting in G0-G1 phase), and surface expression of CD133 in different groups (from 39.3% in the control group to 2.41 in the combination group). Finally, the expression of migration-, and stemness-associated genes were measured by qRT-PCR. We indicated that silencing of CD133 reduces the migration and stemness properties of colorectal cancerous cells. This suppression makes HT-29 cells more sensitive to Oxaliplatin and reduces the effective dose of this chemical drug. Therefore, the suppression of CD133 in combination with Oxaliplatin treatment might be a promising therapeutic approach in the treatment of colorectal cancer.

Astrocytes influence medulloblastoma phenotypes and CD133 surface expression.

The medulloblastoma (MB) microenvironment is diverse, and cell-cell interactions within this milieu is of prime importance. Astrocytes, a major component of the microenvironment, have been shown to impact primary tumor cell phenotypes and metastasis. Based on proximity of MB cells and astrocytes in the brain microenvironment, we investigated whether astrocytes may influence MB cell phenotypes directly. Astrocyte conditioned media (ACM) increased Daoy MB cell invasion, adhesion, and in vivo cellular protrusion formation. ACM conditioning of MB cells also increased CD133 surface expression, a key cancer stem cell marker of MB. Additional neural stem cell markers, Nestin and Oct-4A, were also increased by ACM conditioning, as well as neurosphere formation. By knocking down CD133 using short interfering RNA (siRNA), we showed that ACM upregulated CD133 expression in MB plays an important role in invasion, adhesion and neurosphere formation. Collectively, our data suggests that astrocytes influence MB cell phenotypes by regulating CD133 expression, a key protein with defined roles in MB tumorgenicity and survival.

CD133 in Breast Cancer Cells: More than a Stem Cell Marker.

Initially correlated with hematopoietic precursors, the surface expression of CD133 was also found in epithelial and nonepithelial cells from adult tissues in which it has been associated with a number of biological events. CD133 is expressed in solid tumors as well, including breast cancer, in which most of the studies have been focused on its use as a surface marker for the detection of cells with stem-like properties (i.e., cancer stem cells (CSCs)). Differently with other solid tumors, very limited and in part controversial are the information about the significance of CD133 in breast cancer, the most common malignancy among women in industrialized countries. In this review, we summarize the latest findings about the implication of CD133 in breast tumors, highlighting its role in tumor cells with a triple negative phenotype in which it directly regulates the expression of proteins involved in metastasis and drug resistance. We provide updates about the prognostic role of CD133, underlining its value as an indicator of increased malignancy of both noninvasive and invasive breast tumor cells. The molecular mechanisms at the basis of the regulation of CD133 levels in breast tumors have also been reviewed, highlighting experimental strategies capable to restrain its level that could be taken into account to reduce malignancy and/or to prevent the progression of breast tumors.

Abstract B146: A novel TNIK inhibitor potently downregulates cancer stem cell population through attenuation of Wnt signaling

Abstract Background: TNIK (Traf2- and Nck-interacting kinase) has been identified as a component of the β-catenin and T-cell factor 4 (TCF4) complex that regulates the downstream of Wnt signaling. Recent studies indicated that the Wnt pathway is highly activated in cancer stem cells (CSCs), which have been recognized as the driving force of tumorigenesis and mediating tumor recurrence. Thus, eradicating CSCs by inhibiting Wnt signaling may become a new therapeutic option for a complete cure of cancers. Recently we have demonstrated that TNIK is required for the tumor-initiating function of colorectal CSCs, and therefore TNIK is considered to be attractive targets for drug discovery. We have discovered a thiazole-based TNIK inhibitor having potent inhibitory activity for the Wnt signaling. Further optimization of the thiazole-based TNIK inhibitor resulted in an orally available novel TNIK inhibitor, NC-1. In this study, we evaluated in vivo efficacy of the TNIK inhibitor in mouse models as well as its effects on CSCs in vitro. Materials and Methods: To evaluate the effects of NC-1 on the CSCs in SW620, human colon cancer cells, the enzymatic activity of aldehyde dehydrogenase (ALDH) was analyzed by flow cytometry using an Aldefluor assay. The expression of CSCs surface marker protein, CD133, was analyzed using flow cytometry. Antitumor efficacy of NC-1 was evaluated in a human colorectal tumor xenograft mouse model. Results: We demonstrated that treatment of cancer cells with NC-1 significantly downregulated ALDH positive cell population in a dose-dependent manner. In addition, NC-1 suppressed cell surface expression of CD133 in a dose-dependent manner, suggesting that NC-1 preferentially killed CSCs population in vitro. Furthermore, NC-1 inhibited tumor growth in the HCT116 xenograft mouse model. Citation Format: Yuko Uno, Hideki Moriyama, Shigeki Kashimoto, Mari Masuda, Masaaki Sawa, Tesshi Yamada. A novel TNIK inhibitor potently downregulates cancer stem cell population through attenuation of Wnt signaling [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B146.

Up-modulation of PLC-\u03b22 reduces the number and malignancy of triple-negative breast tumor cells with a CD133+/EpCAM+ phenotype: a promising target for preventing progression of TNBC

BackgroundThe malignant potential of triple negative breast cancer (TNBC) is also dependent on a sub-population of cells with a stem-like phenotype. Among the cancer stem cell markers, CD133 and EpCAM strongly correlate with breast tumor aggressiveness, suggesting that simultaneous targeting of the two surface antigens may be beneficial in treatment of TNBC. Since in TNBC-derived cells we demonstrated that PLC-β2 induces the conversion of CD133high to CD133low cells, here we explored its possible role in down-modulating the expression of both CD133 and EpCAM and, ultimately, in reducing the number of TNBC cells with a stem-like phenotype.MethodsA magnetic step-by-step cell isolation with antibodies directed against CD133 and/or EpCAM was performed on the TNBC-derived MDA-MB-231 cell line. In the same cell model, PLC-β2 was over-expressed or down-modulated and cell proliferation and invasion capability were evaluated by Real-time cell assays. The surface expression of CD133, EpCAM and CD44 in the different experimental conditions were measured by multi-color flow cytometry immunophenotyping.ResultsA CD133+/EpCAM+ sub-population with high proliferation rate and invasion capability is present in the MDA-MB-231 cell line. Over-expression of PLC-β2 in CD133+/EpCAM+ cells reduced the surface expression of both CD133 and EpCAM, as well as proliferation and invasion capability of this cellular subset. On the other hand, the up-modulation of PLC-β2 in the whole MDA-MB-231 cell population reduced the number of cells with a CD44+/CD133+/EpCAM+ stem-like phenotype.ConclusionsSince selective targeting of the cells with the highest aggressive potential may have a great clinical importance for TNBC, the up-modulation of PLC-β2, reducing the number of cells with a stem-like phenotype, may be a promising goal for novel therapies aimed to prevent the progression of aggressive breast tumors.

Overexpression of syndecan-1, MUC-1, and putative stem cell markers in breast cancer leptomeningeal metastasis: a cerebrospinal fluid flow cytometry study

BackgroundCancer is a mosaic of tumor cell subpopulations, where only a minority is responsible for disease recurrence and cancer invasiveness. We focused on one of the most aggressive circulating tumor cells (CTCs) which, from the primitive tumor, spreads to the central nervous system (CNS), evaluating the expression of prognostic and putative cancer stem cell markers in breast cancer (BC) leptomeningeal metastasis (LM).MethodsFlow cytometry immunophenotypic analysis of cerebrospinal fluid (CSF) samples (4.5 ml) was performed in 13 consecutive cases of BCLM. Syndecan-1 (CD138), MUC-1 (CD227) CD45, CD34, and the putative cancer stem cell markers CD15, CD24, CD44, and CD133 surface expression were evaluated on CSF floating tumor cells. The tumor-associated leukocyte population was also characterized.ResultsDespite a low absolute cell number (8 cell/μl, range 1–86), the flow cytometry characterization was successfully conducted in all the samples. Syndecan-1 and MUC-1 overexpression was documented on BC cells in all the samples analyzed; CD44, CD24, CD15, and CD133 in 77%, 75%, 70%, and 45% of cases, respectively. A strong syndecan-1 and MUC-1 expression was also documented by immunohistochemistry on primary breast cancer tissues, performed in four patients. The CSF tumor population was flanked by T lymphocytes, with a different immunophenotype between the CSF and peripheral blood samples (P ≤ 0.02).ConclusionsFlow cytometry can be successfully employed for solid tumor LM characterization even in CSF samples with low cell count. This in vivo study documents that CSF floating BC cells overexpress prognostic and putative cancer stem cell biomarkers related to tumor invasiveness, potentially representing a molecular target for circulating tumor cell detection and LM treatment monitoring, as well as a primary target for innovative treatment strategies. The T lymphocyte infiltration, documented in all CSF samples, suggests a possible involvement of the CNS lymphatic system in both lymphoid and cancer cell migration into and out of the meninges, supporting the extension of a new form of cellular immunotherapy to LM. Due to the small number of cases, validation on large cohorts of patients are warranted to confirm these findings and to evaluate the impact and value of these results for diagnosis and management of LM.

Cancer stem cells CD133 inhibition and cytotoxicity of certain 3-phenylthiazolo[3,2-a]benzimidazoles: design, direct synthesis, crystal study and in vitro biological evaluation

Cancer stem cells (CSCs) have been objects of intensive study since their identification in 1994. Adopting a structural rigidification approach, a novel series of 3-phenylthiazolo[3,2-a]benzimidazoles 4a–d was designed and synthesised, in an attempt to develop potent anticancer agent that can target the bulk of tumour cells and CSCs. The anti-proliferative activity of the synthesised compounds was evaluated against two cell lines, namely; colon cancer HT-29 and triple negative breast cancer MDA-MB-468 cell lines. Also, their inhibitory activity against the cell surface expression of CD133 was examined. In particular, compound 4b emerged as a promising hit molecule as it manifested good antineoplastic potency against both tested cell lines (IC50 = 9 and 12 μM, respectively), beside its ability to inhibit the cell surface expression of CD133 by 50% suggesting a promising potential of effectively controlling the tumour by eradicating the tumour bulk and inhibiting the proliferation of the CSCs. Moreover, compounds 4a and 4c showed moderate activity against HT-29 (IC50 = 21 and 29 μM, respectively) and MDA-MB-468 (IC50 = 23 and 24 μM, respectively) cell lines, while they inhibited the CD133 expression by 14% and 48%, respectively. Finally, a single crystal X-ray diffraction was recorded for compound 4d.

Etoposide-Bevacizumab a new strategy against human melanoma cells expressing stem-like traits.

Tumors contain a sub-population of self-renewing and expanding cells known as cancer stem cells (CSCs). Putative CSCs were isolated from human melanoma cells of a different aggressiveness, Hs294T and A375 cell lines, grown under hypoxia using “sphere-forming assay”, CD133 surface expression and migration ability. We found that a cell sub-population enriched for P1 sphere-initiating ability and CD133 expression also express larger amount of VEGF-R2. Etoposide does not influence phenotype of this sub-population of melanoma cells, while a combined treatment with Etoposide and Bevacizumab significantly abolished P1 sphere-forming ability, an effect associated with apoptosis of this subset of cells. Hypoxic melanoma cells sorted for VEGF-R2/CD133 positivity also undergo apoptosis when exposed to Etoposide and Bevacizumab. When Etoposide and Bevacizumab-treated hypoxic cells were injected intravenously into immunodeficient mice revealed a reduced capacity to induce lung colonies, which also appear with a longer latency period. Hence, our study indicates that a combined exposure to Etoposide and Bevacizumab targets melanoma cells endowed with stem-like properties and might be considered a novel approach to treat cancer-initiating cells.

Heterodimeric Bispecific Single Chain Variable Fragments (scFv) Killer Engagers (BiKEs) Enhance NK-cell Activity Against CD133+ Colorectal Cancer Cells.

Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse, and express the well-known surface marker CD133. DNA fragments from two fully humanized single chain fragment variable (scFv) antibodies recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigens to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated a unique antibody that recognized the molecular framework and was species cross-reactive. In vitro chromium-51 ((51)Cr) release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5% CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133 and that this anti-CD133 scFv may be useful in this bispecific platform or perhaps in the design of more complex trispecific molecules for carcinoma therapy.

2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones: Synthesis, crystal study and cancer stem cells CD133 targeting potential

In order to develop a potent anti-tumor agent that can target both cancer stem cells and the bulk of tumor cells, a series of 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o was synthesized. All compounds were evaluated for their anti-proliferative activity towards colon HT-29 cancer cell line. In addition, their inhibitory effect against cell surface expression of CD133, a potent cancer stem cells (CSCs) marker, in the same cells was evaluated by flow cytometry at 10 μM. Compound 5l emerged as the most active anti-proliferative analog against HT-29 (IC50 = 18.83 ± 1.37 μM), that almost equipotent as 5-fluorouracil (IC50 = 15.83 ± 1.63 μM) with 50.11 ± 4.05% inhibition effect on CD133 expression, suggested dual targeted effect. Also, compounds 5h, 5j, 5k and 5m-o inhibited the expression of CD133 with more than 50%. The SAR study pointed out the significance of substitution of the pendent phenyl group with lipophilic electron-donating groups or replacing it by 2-thienyl or 2-furyl groups.

Abstract 748: The glucose metabolism targeting therapies and withaferin A eliminate epidermal growth factor tyrosine kinase inhibitor-induced drug-tolerant persisters in non-small lung cancer cells

Abstract In pathway-targeted cancer drug therapies, the relatively rapid emergence of drug-tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. The aim of this study is (1) to clarify the subpopulations of DTPs in EGFR mutated non-small lung cancer (NSCLC) cells that have different roles in the resistance to epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) and (2) to develop effective treatments. In this study, we used two EGFR mutated lung adenocarcinoma cell lines, PC9 and II-18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 month. We analyzed the mRNA expression of the stem cell related markers by qRT-PCR and the expression of the cellular senescence associated proteins by Western blotting. Then, we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by the glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A. The time-dependent upregulation of the stem cell-related markers was observed until day 24. Western blot analysis revealed that the cellular senescence associated proteins were upregulated in a similar time course. Senescent cells secrete various inflammatory cytokines and chemokines, a phenomenon known as SASP. Recently, SASP has been reported to contribute to the emergence and maintenance of cancer stem-like cells. Using FACS analysis, DTPs were divided into the two populations depending on the surface expression of CD133. CD133high DTPs were characterized by the higher expression of stem cell related markers. On the other hand, CD133low DTPs were characterized by higher expression of the cellular senescence associated proteins. Rapamycin is reported to suppress SASP. Gefitnib and rapamycin effectively suppressed the emergence of CD133high DTPs. We found CD133low DTPs showed higher glucose metabolic state, consistent with the previous reports that show senescence-associated metabolic reprogramming is induced by anticancer therapies. We targeted this metabolic change using a glucose transporter inhibitor (phloretin) and a glycolytic blocker (2DG). These agents successfully suppressed the emergence of CD133high DTPs. Finally, withaferin A could inhibit the emergence of CD133high DTPs. In conclusion, we found that the EGFR-TKI-induced DTPs in EGFR mutated NSCLC cells were composed of at least two distinct cell populations: CD133high DTPs with stem-like characters and CD133low DTPs with cellular senescent characters. CD133low DTPs supported CD133high DTPs via SASP and blocking SASP effectively suppressed the emergence of CD133high DTPs. Furthermore, our results suggest that the glucose metabolism targeting therapies and withaferin A can be new therapeutic candidates to ablate DTPs. Citation Format: Kei Kunimasa, Tatsuya Nagano, Yohei Shimono, Shuntaro Tokunaga, Daisuke Tamura, Motoko Tachihara, Kazuyuki Kobayashi, Yoshihiro Nishimura. The glucose metabolism targeting therapies and withaferin A eliminate epidermal growth factor tyrosine kinase inhibitor-induced drug-tolerant persisters in non-small lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 748. doi:10.1158/1538-7445.AM2015-748

Selective lentiviral gene delivery to CD133-expressing human glioblastoma stem cells.

Glioblastoma multiforme (GBM) is a deadly primary brain malignancy. Glioblastoma stem cells (GSC), which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance.

Correlation between the surface expression of CD133 and the phenotype of breast tumor cells

Originally considered a marker of hematopoietic stem cells, CD133/prominin is a highly glycosylated trans-membrane protein expressed in various tissues, such as breast, in which it seems to regulate ductal branching but not regenerative capacity [1]. CD133 is also expressed in various solid tumors, including breast cancer, in which CD133-positivity seems to identify a restricted subgroup of tumor stem cells [2]. CD133 expression was heterogeneous in different breast carcinomas but, in triple- negative (ER-, PR-, HER2-) invasive ductal breast carcinoma, CD133 correlates with tumor size, metastasis and clinical stage [3]. In order to establish a correlation between the surface recognition of CD133 and the phenotype of tumor cells, the highly invasive breast-derived MDA-MB-231 cells (ER-, PR-, HER2-) were subjected to immunomagnetic separation of CD133+ and CD133- subpopulations, which were analyzed for malignant properties. In comparison to CD133- cells, the expression of CD133 characterizes cells with a larger adhesion area, lower proliferation rate and reduced migration speed. This phenotype correlates with altered expression of malignancy-associated proteins and with a peculiar pattern of PLC, in turn involved in proliferation and motility of breast tumor cells (4-6). This suggests that, in triple negative ductal breast tumor-derived cells, the expression of CD133 characterizes a small subset of cells with a less undifferentiated phenotype. The reduced expression of CD133 at membrane level may constitute a marker of the switch of tumor cells from a less malignant to a mature phenotype since it correlates with the de-regulation of proteins involved in cell proliferation, motility and invasion.

CD133+gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity

To identify cancer stem cells (CSCs) in human gallbladder carcinomas (GBCs). Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of the sphere-forming cells, including self-renewal, differentiation potential, chemoresistance and tumorigenicity, were determined in vitro or in vivo. Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry. The sphere-colony-formation ability and tumorigenicity of CD133(+) cells were assayed. In vitro culture experiments revealed that floating spheroids were generated from primary GBC cells, and these sphere-forming cells could generate new progeny spheroids in serum-free media. Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4, Nanog, and nestin (P < 0.05). The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells (P < 0.05). Spheroid cells were more resistant to chemotherapeutic reagents than the congenetic adherent cells (P < 0.05). Flow cytometry showed enriched CD133(+) population in sphere-forming cells (P < 0.05). CD133(+) cells possessed high colony-formation ability than the CD133(-) population (P < 0.01). CD133(+) cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts (P < 0.05). CD133 may be a cell surface marker for CSCs in GBC.

Activation of the Protective Arm of Renin Angiotensin System (RAS) Corrects the Reparative Dysfunction of Diabetic CD34+ Cells.

AbstractAbstract 2637 Purpose:RAS plays a vital role in regulating many physiological processes of the vascular system. Angiotensin II (Ang II), a product of angiotensin converting enzyme (ACE), mediates its effects through activation of either the AT1 receptor – to induce vasoconstriction, proliferation, fibrosis, and inflammation – or the AT2 receptor to promote NO generation. The protective arm of RAS involves ACE2, which produces angiotensin-(1-7) [Ang-(1-7)]. Ang-(1-7) activates the MAS receptor to promote vascular health. Because diabetic endothelial progenitor cells are dysfunctional and this limits their utility in autologous cell therapy, we asked whether angiotensin (Ang)-(1-7) could restore the vasoreparative function of diabetic CD34+cells. Methods:Healthy nondiabetic (ND) and diabetic (D) Lin−CD45midCD34+ cells were obtained from peripheral blood mononuclear cells (PB-MNCs) by FACS. The effect of Ang-(1-7) on migration, proliferation, NO bioavailability, reactive oxygen species (ROS) levels and NADPH oxidase activity were evaluated in ND- and D-CD34+ cells. The effect of Ang-(1-7) on the formation of ECFCs from ND- and D-MNCs was evaluated. Ang-(1-7) production by cells was analyzed and the expression of ACE2 and Mas-receptor were assessed by real-time PCR and flow cytometry. Effects of ACE2 activators XNT and DIZE in CD34+ cells were also evaluated. D-CD34+ cells were genetically modified to overexpress Ang-(1-7) by lentiviral Ang-(1-7)-fusion transgene and their function was evaluated in vitro. The effect of transduction on the surface expression of CD133, CD34 and CD309 was assessed. In vivo homing function was assessed in a mouse model of ischemia-reperfusion (I/R). After one week of I/R insult, when retinal capillary damage was appreciable, CD34 cells were intravitreally injected. Neural retinas were harvested after 48 hours and human cells within the mouse vasculature were localized by immunohistochemistry. Results:Migration to SDF1- and VEGF-were impaired in D-CD34+ cells. In contrast, Ang-(1-7)-induced migration in both D-CD34+ and ND-CD34+cells was dependent on Mas receptor expression and eNOS. ROS levels and NADPH oxidase activity were reduced and proliferation and NO bioavailability were restored in D-CD34+ cells by Ang-(1-7). ECFCs from D-MNCs were appeared only in the presence of Ang-(1-7). Migration, NO release and Ang-(1-7) release by ACE2 activators, XNT and DIZE, were significantly decreased in D-CD34+ cells. Ang-(1-7) release and ACE2 expression were decreased in D-cells while Mas-receptor expression was similar that in ND-cells. Ang-(1-7) gene-modified cells showed reduced ROS levels, increased NO bioavailability, enhanced migration to SDF-1 and proliferation. Lentiviral transduction did not alter surface expression of CD133, CD34 and CD309. Ang-(1-7)-overexpression restored the homing efficiency of D-CD34+ cells similar to that of ND-CD34+ cells in vivo. Conclusions:Ang-(1-7) stimulates the vasoreparative functions in CD34+ cells. Ang-(1-7) bypasses the reduced ACE2 seen in diabetic CD34+ cells and restores the vasoreparative potential of these cells by decreasing oxidative stress and normalizing NO bioavailability. Pharmacological strategies that either increase ACE2 or Ang-(1-7) in diabetic CD34+ cells will improve their therapeutic utility for autologous cell therapy in treatment of diabetic complications. Disclosures:No relevant conflicts of interest to declare

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.