scholarly journals 3357 TL1 Team Approach to Using a Combination of Ganglioside 2 and 3 as an Immunoaffinity Target for Circulating Osteosarcoma Cell Detection

2019 ◽  
Vol 3 (s1) ◽  
pp. 134-135
Author(s):  
Henrietta Fasanya ◽  
Pablo Joaquin Dopico ◽  
Zachary J. Yeager ◽  
Hugh Fan ◽  
Dietmar W. Siemann
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Cell Lines ◽  
Treatment Efficacy ◽  
Capture Efficiency ◽  
Cell Detection ◽  
Surface Markers ◽  
Osteosarcoma Cell ◽  
Cell Surface Markers ◽  
Osteosarcoma Cells

OBJECTIVES/SPECIFIC AIMS: The objective of our collaboration is to develop a strong trans-disciplinary team consisting of microfluidics engineers, cancer biologists, and clinicians, to identify a universal marker to detect circulating osteosarcoma cells (COC) using microfluidic devices. Our goals are 3 fold: 1) Identify cell surface markers unique to osteosarcoma (OS) for COC isolation, 2) Develop a Geometrically Enhanced Mixing (GEM) device to isolate COCs, and 3) Evaluate the efficacy of GEM device to detect COCs in patients with OS. The long term goal of this collaboration is to utilize this cell detection approach to evaluate treatment efficacy and correlate the presence of circulating osteosarcoma cells with metastatic incidence. METHODS/STUDY POPULATION: In this phase of our study, we have identified an abundant and conserved cell surface marker across a panel of OS cell lines. Flow cytometry was used to evaluate the relative expression of Epithelial Cell Adhesion Molecule (EpCAM), and Ganglioside 2 or/and 3 (GD2/3) on a panel of OS cell lines. An antibody coated GEM microfluidic device is used to affirm the efficacy of GD2/3 to capture COCs. Further capture studies will be conducted using OS cell spiked blood samples. Analysis of variance (ANOVA) will be used to determine any significant difference in capture efficiency between EpCAM, GD2/3 cell surface markers. RESULTS/ANTICIPATED RESULTS: Our results demonstrate that EpCAM is not a suitable marker for COC detection. Results from our flow cytometry studies demonstrate that GD2/3 expression is significantly higher than EpCAM expression, across all OS cell lines within our panel. The cell capture efficiency strongly correlates with the cell surface expression data obtained from flow cytometry analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: OS is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. At diagnosis, 80% of patients will present with metastasis, however only 20% of these cases are clinically detectable. Innovative strategies to identify patients at risk of metastasis would allow for stratification of intervention therapies. Liquid biopsies are a novel alternative to current diagnostic imaging systems to monitor metastatic incidence and treatment efficacy. The detection of circulating tumor cells (CTCs) through routine blood sampling has the potential to be used clinically for earlier detection, monitoring the treatment of metastatic cancers and surveying the effect of therapeutic interventions on metastasis. To date, the majority of the studies on CTCs have evaluated their presence in carcinomas. Although sarcomas are rare, they generally have a poorer prognosis. This study will address one of the unmet medical needs in the field of CTC detection; the identification of cell surface OS makers to improve binding specificity, increase purity, and maintain a high capture efficiency.

scholarly journals 2090 TL1 team approach to osteosarcoma cell detection

2018 ◽  
Vol 2 (S1) ◽  
pp. 33-33
Author(s):  
Pablo J. Dopico ◽  
Henrietta Fasanya ◽  
Dietmar W. Siemann ◽  
Hugh Z. Fan
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Diagnostic Imaging ◽  
Cell Lines ◽  
Microfluidic Device ◽  
Capture Efficiency ◽  
Clinical Samples ◽  
Cell Detection ◽  
Surface Markers ◽  
Cell Surface Markers

OBJECTIVES/SPECIFIC AIMS: The objective of our collaboration is to develop a strong transdisciplinary team consisting of microfluidics engineers, cancer biologists, and clinicians, to identify cell surface markers capable of detecting circulating osteosarcoma cells (COC) using microfluidic devices. Our goals are 3-fold: (1) Identify cell surface markers unique to osteosarcoma (OS) for COC isolation, (2) develop a Geometrically Enhanced Mixing (GEM) device to isolate COCs, and (3) Evaluate the efficacy of GEM device to detect COCs in OS patients under treatment. The long-term goal is to utilize this cell detection approach to correlate the presence of COC with metastatic incidence. METHODS/STUDY POPULATION: To identify a marker to capture COCs we are utilizing flow cytometry and microfluidic capture devices. Flow cytometry will be used to evaluate the relative expression of epithelial cell adhesion molecule (EpCAM), CD45, cell surface vimentin (CSV), insulin-like growth factor 2 (IGF2R), interleukin 11 receptor subunit alpha (IL-11Rɑ), ganglioside 2 (GD2), and receptor activator of nuclear factor κ-B (RANK) on a panel of OS cell lines. These cell surface markers were selected based on an extensive review of OS cell surface markers. OS cell capture efficacy will be assessed by passaging a known concentration of OS cells through a GEM microfluidic device coated with antibodies targeting the selected marker, as indicated by flow cytometry. Once captured, COCs on the device will be analyzed and the capture efficiency for the indicated marker will be measured. ANOVA will be used to determine any significant difference in capture efficiency between marker types. Once an optimal marker or panel of markers has been selected we will conduct capture studies using OS cell spiked blood samples followed by clinical samples obtained from OS patients. In clinical samples, COC detection will be validated using the FDA approved triple immunocytochemistry technical definition of a circulating tumor cell (CTC). This will enable COCs to be differentiated from the normal whole blood cell population by selecting for CD45−, EpCAM+, and cytokeratin+ cells. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown that on our microfluidic device, EpCAM, a marker commonly used to identify circulating tumor cells in other cancer settings, has a poor capture efficiency (15.9%+7.7%) for HU09 OS cells while the same setup with EpCAM has a capture efficiency of 56.9%+2.7% for BXPc-3 pancreatic cells. We therefore anticipate our flow cytometry studies to show a low expression of EpCAM and CD45 for OS cell lines, while showing a moderate to high expression of CSV, IGF2R, IL-11Rɑ, GD2, and RANK. We expect to show a 60%–80% capture efficiency for markers selected for COC capture. Currently, CSV and GD2 are particularly promising as markers based on previously published studies. DISCUSSION/SIGNIFICANCE OF IMPACT: OS is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. At diagnosis 80% of patients will present with metastasis, however only 20% of these cases are clinically detectable. Innovative strategies to identify patients at risk of metastasis would allow for stratification of intervention therapies. Currently, tumor recurrence and metastasis are primarily dependent on diagnostic-imaging modalities such as computerized tomography or positron emission tomography scans. Unfortunately, these imaging modalities can only detect tumor masses of significant size (106 tumor cells). Liquid biopsies are a novel alternative to current diagnostic imaging systems to monitor metastatic incidence and treatment efficacy. The detection of CTCs through routine blood sampling has the potential to be used clinically for earlier detection, monitoring the treatment of metastatic cancers and surveying the effect of therapeutic interventions on metastasis. To date, the majority of the studies on CTCs have evaluated their presence in carcinomas. Although sarcomas are rare, they generally have a poor prognosis. This study will address one of the unmet medical needs in the field of CTC detection; the identification of cell surface OS makers to improve binding specificity, increase purity, and maintain a high capture efficiency. This phase of our proposal will evaluate the most abundant and conserved markers across a panel of OS cell lines. Once a marker or panel of markers is selected, we will begin to develop a microfluidic device that can be used clinically to detect CTCs in this disease setting.

scholarly journals MicroRNA-19 contributes to the malignant phenotypes of osteosarcoma in vitro by targeting Pax6

Tumor Biology ◽  
2018 ◽  
Vol 40 (1) ◽  
pp. 101042831774470 ◽  
Author(s):  
Qingbing Meng ◽  
Ming Dai ◽  
Xuejun Nie ◽  
Wensheng Zhang ◽  
Xingli Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Biological Behavior ◽  
Luciferase Reporter ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Transwell Assay ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Diphenyltetrazolium Bromide

This study was conducted to detect the expression of miR-19 and Pax6 (Paired box protein 6) in human osteosarcoma cells and the effects on biological characteristics of osteosarcoma cells. Quantitative real-time polymerase chain reaction was used to detect the expression of Pax6 and miR-19 in normal human osteoblasts (hFOB 1.19) and osteosarcoma cell lines (U2OS, Saos-2, and MG-63). Results showed that miR-19 was significantly upregulated in osteosarcoma cell lines compared with that in hFOB 1.19 cells, while the expression of Pax6 messenger RNA was significantly downregulated. Pax6 was defined as the target gene of miR-19 which was validated by luciferase reporter gene analysis. Results indicated that miR-19 had an interaction with Pax6 3′-untranslated region. At the same time, the protein expression of Pax6 was significantly decreased in the MG-63 cells transfected with miR-19 mimic and was notably enhanced in osteosarcoma MG-63 cells transfected with miR-19 inhibitor. These data suggested that Pax6 was a target of miR-19 in osteosarcoma MG-63 cells. The effects of miR-19 on the biological behavior of MG-63 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Transwell assay. Results showed that the downregulation of miR-19 inhibited cell viability, reduced the percentage of cells in S phase and the number of cells passing through the Transwell chamber, and increased the number of apoptotic cells. Western blot analysis showed that the inhibition of miR-19 significantly increased the expression of epithelial proteins (E-cadherin and β-catenin) and decreased the expression of mesenchymal protein (Vimentin), extracellular signal–regulated kinase, and phosphorylated extracellular signal–regulated kinase in MG-63 cells. MiR-19 inhibitor and Pax6 small interfering RNA were simultaneously transfected into MG-63 cells. Results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Transwell assay demonstrated that the inhibition of Pax6 expression in MG-63 cells could reverse the cell biological effects induced by the inhibition of miR-19 expression. Based on these findings, it was suggested that miR-19, upregulated in osteosarcoma cells, negatively regulated the expression of Pax6, which can promote the malignant phenotypes of osteosarcoma cells via activation of the extracellular signal–regulated kinase signaling pathways. Therefore, miR-19/Pax6 may offer potential for use as a target for the treatment of osteosarcoma.

Flow cytometry and live cell imaging for characterization of cell surface markers on human tissue-derived progenitors

Cytotherapy ◽  
2020 ◽  
Vol 22 (5) ◽  
pp. S69-S70
Author(s):  
W.A. Bova ◽  
V.R. Mantripragada ◽  
V. Luangphakdy ◽  
G.F. Muschler
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Human Tissue ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Surface Markers ◽  
Cell Surface Markers

Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements

1988 ◽  
Vol 8 (12) ◽  
pp. 5358-5368
Author(s):  
C F Barth ◽  
E H Humphries
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Light Chain ◽  
Lymphoid Cells ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Chain Locus ◽  
Light Chain Locus

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.

scholarly journals Gingival-Derived Mesenchymal Stem Cell from Rabbit (Oryctolagus cuniculus): Isolation, Culture, and Characterization

2020 ◽  
Author(s):  
Alexander Patera Nugraha ◽  
Fedik Abdul Rantam ◽  
Ida Bagus Narmada ◽  
Diah Savitri Ernawati ◽  
Igo Syaiful Ihsan
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Surface ◽  
Osteogenic Differentiation ◽  
Oryctolagus Cuniculus ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Alizarin Red ◽  
Regenerative Dentistry

Abstract Objective This study aims to confirm whether the GDMSCs isolated from rabbit’s (Oryctolagus cuniculus) gingiva are mesenchymal stem cells (MSCs). Materials and Methods This study design was partly quasi-experimental with an observational design. GDMSCs were isolated from the gingiva of healthy male rabbits (O. cuniculus) (n = 2), 6 months old, and 3 to 5 kg of body weight. The specific cell surface markers of MSCs; clusters of differentiation (CD), namely, CD44, CD73, CD90, CD105, and CD200 expressions; and hematopoietic stem cell surface markers CD34 and CD45 were examined using flow cytometry and immunohistochemistry with immunofluorescence. The osteogenic differentiation of isolated GDMSCs was examined using alizarin red staining. Results GDMSCs in the fourth passage showed a spindle-like formation and fibroblast-like cells that attached to the base of the culture plate. GDMSCs were MSCs that positively expressed CD44, CD73, CD90, CD105, and CD200 but did not express CD34 and CD45 when examined using flow cytometry and immunohistochemical analysis. GDMSCs had osteogenic differentiation confirmed by calcified deposits in vitro with a red–violet and brownish color after alizarin red staining. Conclusion GDMSCs isolated from the rabbits (O. cuniculus) were confirmed as MSCs in vitro documented using immunohistochemistry and flow cytometry. GDMSCs can differentiate into osteogenic lineage in vitro that may be suitable for regenerative dentistry.

Chromosomes and cell surface markers of marmoset lymphocytes and Epstein-Barr virus-transformed marmoset cell lines

1981 ◽  
Vol 3 (2) ◽  
pp. 101-108 ◽  
Author(s):  
Donald R. Johnson ◽  
Göran Levan ◽  
George Klein ◽  
Stephen M. Nigida ◽  
Lauren G. Wolfe
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer

2018 ◽  
Vol 115 (19) ◽  
pp. E4473-E4482 ◽  
Author(s):  
John K. Lee ◽  
Nathanael J. Bangayan ◽  
Timothy Chai ◽  
Bryan A. Smith ◽  
Tiffany E. Pariva ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Surface ◽  
Cell Lines ◽  
Treatment Resistance ◽  
Specific Cell ◽  
Surface Markers ◽  
Mrna Levels ◽  
Cell Surface Markers ◽  
Cell Surface Antigens ◽  
Cancer Subtypes

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.

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