Journal of the National Cancer Institute Advance Access published online on February 24, 2009
JNCI Journal of the National Cancer Institute, doi:10.1093/jnci/djn509
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© The Author 2009. Published by Oxford University Press.
ARTICLES |
In Vivo Imaging, Tracking, and Targeting of Cancer Stem Cells
Affiliations of authors: Division of Molecular and Cellular Oncology, Department of Radiation Oncology (EV, KK, CL, LDD, JTM, WM, FP), Department of Anesthesiology (ME, ES), David Geffen School of Medicine at University of California, Los Angeles, Biostatistics and Radiological Sciences, School of Public Health (JWS), University of California, Los Angeles, Jonsson Comprehensive Cancer Center (WM, FP), University of California, Los Angeles
Correspondence to: Frank Pajonk, MD, PhD, Division of Molecular and Cellular Oncology, Department of Radiation Oncology, David Geffen School of Medicine at University of California, Los Angeles, 10833 Le Conte Ave, Los Angeles, CA 90095-1714 (e-mail: fpajonk{at}mednet.ucla.edu).
Background: There is increasing evidence that solid cancers contain cancer-initiating cells (CICs) that are capable of regenerating a tumor that has been surgically removed and/or treated with chemotherapy and/or radiation therapy. Currently, cell surface markers, like CD133 or CD44, are used to identify CICs in vitro; however, these markers cannot be used to identify and track CICs in vivo. The 26S proteasome is the main regulator of many processes within a proliferating cell, and its activity may be altered depending on the phenotype of a cell.
Methods: Human glioma and breast cancer cells were engineered to stably express ZsGreen fused to the carboxyl-terminal degron of ornithine decarboxylase, resulting in a fluorescent fusion protein that accumulates in cells in the absence of 26S proteasome activity; activities of individual proteases were monitored in a plate reader by detecting the cleavage of fluorogenic peptide substrates. Proteasome subunit expression in cells expressing the fusion protein was assessed by quantitative reverse transcription—polymerase chain reaction, and the stem cell phenotype of CICs was assessed by a sphere formation assay, by immunohistochemical staining for known stem cell markers in vitro, and by analyzing their tumorigenicity in vivo. CICs were tracked by in vivo fluorescence imaging after radiation treatment of tumor-bearing mice and targeted specifically via a thymidine kinase–degron fusion construct. All P values were derived from two-sided tests.
Results: Cancer cells grown as sphere cultures in conditions, which enrich for cancer stem cells (CSCs), had decreased proteasome activity relative to the respective monolayers (percent decrease in chymotryptic-like activity of sphere cultures relative to monolayers—U87MG: 26.64%, 95% confidence interval [CI] = 10.19 to 43.10, GL261, 52.91%, 95% CI = 28.38 to 77.43). The cancer cells with low proteasome activity can thus be monitored in vitro and in vivo by the accumulation of a fluorescent protein (ZsGreen) fused to a degron that targets it for 26S proteasome degradation. In vitro, ZsGreen-positive cells had increased sphere-forming capacity, expressed CSC markers, and lacked differentiation markers compared with ZsGreen-negative cells. In vivo, ZsGreen-positive cells were approximately 100-fold more tumorigenic than ZsGreen-negative cells when injected into nude mice (ZsGreen positive, 30 mice per group; ZsGreen negative, 31 mice per group), and the number of CICs in tumors increased after 72 hours post radiation treatment. CICs were selectively targeted via a proteasome-dependent suicide gene, and their elimination in vivo led to tumor regression.
Conclusion: Our results demonstrate that reduced 26S proteasome activity is a general feature of CICs that can easily be exploited to identify, track, and target them in vitro and in vivo.
| Context and Caveats Prior knowledge It had become evident that in many solid cancers there are small subpopulations of cells with stem cell–like properties known as cancer initiating cells (CICs) (or cancer stem cells [CSCs]) that are relatively resistant to conventional cancer therapies. Methods to identify and track these cells in vivo were lacking. Study design Cancer cells were engineered to express a fluorescent protein that is a target of the 26S protesome, a multiprotein complex which appeared to have reduced proteolytic activity in CICs. The correlation of various CIC/CSC phenotypes in the engineered cells with fluorescence, and thus 26S proteosome activity, was assessed. Contribution This study found that reduced 26S proteosome activity was closely correlated with CIC phenotypes in glioma and breast cancer cells. Thus, engineering cells to express a substrate of the protease is a viable method to identify and track these cell populations in vivo. Implications The ability to identify and track CICs in animal models of cancer may allow better assessment of therapeutic approaches compared to conventional methods such as measuring tumor response. Limitations The CICs with low protease activity may themselves be a heterogenous population of cells that needs to be further defined. From the Editors
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Manuscript received July 3, 2008; revised November 24, 2008; accepted December 31, 2008.
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J Natl Cancer Inst 2009 101: 281.