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Sigma-2 Receptor as Potential Indicator of Stem Cell Differentiation

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Molecular Imaging and Biology Aims and scope Submit manuscript

Abstract

Purpose

The sigma-2 (σ2) receptor is a potential biomarker of proliferative status of solid tumors. Specific synthetic probes using N-substituted-9-azabicyclo [3.3.1]nonan-3α-yl carbamate analogs have been designed and implemented for experimental cancer diagnosis and therapy.

Procedures

We employed the fluorescently labeled σ2 receptor probe, SW120, to evaluate σ2 receptor expression in human stem cells (SC), including: bone marrow stromal, neural progenitor, amniotic fluid, hematopoetic, and embryonic stem cells. We concurrently evaluated the intensity of SW120 and 5-ethynyl-2′-deoxyuridine (EdU) relative to passage number and multi-potency.

Results

We substantiated significantly higher σ2 receptor density among proliferating SC relative to lineage-restricted cell types. Additionally, cellular internalization of the σ2 receptor in SC was consistent with receptor-mediated endocytosis and confocal microscopy indicated SW120 specific co-localization with a fluorescent marker of lysosomes in all SC imaged.

Conclusion

These results suggest that σ2 receptors may serve to monitor stem cell differentiation in future experimental studies.

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Abbreviations

HSC:

Hematopoietic stem cell

ESC:

Embryonic stem cell

BMSC:

Bone marrow stromal cell

NPC:

Neural progenitor cell

AFSC:

Amniotic fluid stem cell

CFU:

Colony-forming unit

CFC:

Colony-forming conditions

EdU:

5-ethynyl-2′-deoxyuridine

Ki-67:

Antigen Ki-67 cellular marker of proliferation

PAO:

Phenylarsine oxide

MFI:

Mean fluorescence intensity

σ2 receptor :

Sigma-2 receptor

NBD:

7-Nitrobenzo-2-oxa-1,3-diazole

SW120:

NBD-labeled σ2-receptor -specific ligand

SV119:

Unconjugated σ2 receptor ligand

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Acknowledgment

This work was supported by the Intramural Research Program in the Clinical Center at the National Institutes of Health.

Conflict of interest

The σ2 receptor ligands described in this paper have been licensed from Washington University (RH Mach, inventor) by Isotrace Technologies, Inc., St Charles, MO.

Author information

Authors and Affiliations

  1. Frank Laboratory, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, Clinical Center, Bldg. 10, Room BIN256, Bethesda, MD, 20892, USA

    Jodi L. Haller, Aneeka Chaudhry & Joseph A. Frank

  2. Neumann Laboratory, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, Clinical Center, Bldg. 10, Room BIN256, Bethesda, MD, 20892, USA

    Irina Panyutin

  3. Department of Radiology, Division of Radiological Sciences, Washington University School of Medicine, St. Louis, MO, USA

    Chenbo Zeng & Robert H. Mach

  4. National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, USA

    Joseph A. Frank

Authors
  1. Jodi L. Haller
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  2. Irina Panyutin
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  3. Aneeka Chaudhry
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  4. Chenbo Zeng
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  5. Robert H. Mach
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Corresponding author

Correspondence to Jodi L. Haller.

Electronic Supplementary Material

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ESM 1

Fig. S1. Phenotypic changes upon long term in vitro culture and differentiation of BMSC. a BMSC passage 2, b BMSC passage 25, c Oil red O positive BMSC following 14 day adipogenesis. Scale bars, 100 μm. Fig. S2. Apoptosis noted upon σ2 receptor binding in tumor cells not evident in BMSC or AFSC. a BMSC, AFSC, NPC, and comparative tumor cell line C6 glioma were incubated with annexin V-Alexa 647 only 15 min, b BMSC, AFSC, NPC, and C6 glioma incubated SW120 10 nmol/L, 30 min, followed by annexinV-Alexa 647, 15 min, c 48-h serum starvation of BMSC, AFSC, NPC and C6 glioma, followed by annexinV-Alexa 647, 15 min, and propidium iodide (10 μg/mL) staining. Bars indicate level fluorescence intensity of unstained cells. Fluorescence detection as follows: FL1-A: SW120, FL2-A: propidium iodide, FL4-A: annexin V-Alexa 647. All samples evaluated with flow cytometry, experiment done in triplicate, values normalized to MFI unstained cells in annexin V binding buffer. Fig. S3. Molecular structures of additional Sigma Receptor-specific ligands. a SV-119, unconjugated σ2 receptor ligand and b (+) pentazocine, σ1 receptor ligand. Fig. S4. Ki-67 biomarker of proliferative status demonstrated similar activity to SW120. Percentage of SW120+ cells in population (white), percentage SW120+/Ki-67 double-positive same population (gray). * denotes BMSC under 14 day adipogenic conditions and MDA-MB435 has been included as reference tumor cell line. Flow cytometric analysis identified the SW120 positive cellular fractions across the indicated SC types as almost exclusively Ki-67 antigen positive. All samples evaluated in triplicate with values normalized to MFI of unstained cells in stain buffer. (PDF 1992 kb)

Table S1

Summary of the assays performed for SC in this study. Not all procedures could be carried out on ESC and HSC because of limited availability of the cell products. *NPC differentiation was performed in chamber slides for the purpose of confocal microscopy SW120/Lysotracker assay only. Normal human astrocyte cultures were substituted for differentiating NPC to astrocytes and were obtained from Lonza (Walkerville, MD) for flow cytometry analysis. HSC differentiation was implemented for flow cytometry assay under semi-solid colony-forming conditions not suitable for confocal imaging. (DOC 35 kb)

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Haller, J.L., Panyutin, I., Chaudhry, A. et al. Sigma-2 Receptor as Potential Indicator of Stem Cell Differentiation. Mol Imaging Biol 14, 325–335 (2012). https://doi.org/10.1007/s11307-011-0493-3

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  • DOI: https://doi.org/10.1007/s11307-011-0493-3

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