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Fluorescent tumour imaging of type I IGF receptor in vivo: comparison of antibody-conjugated quantum dots and small-molecule fluorophore.

Zhang H, Zeng X, Li Q, Gaillard-Kelly M, Wagner CR, Yee D - Br. J. Cancer (2009)

Bottom Line: After conjugation with AVE-1642, a humanised anti-IGF1R monoclonal antibody, both QDs (705 nm) or Alexa 680 (small-molecule fluorophore) detected expression and downregulation of IGF1R in vitro.Depletion of macrophages by clodronate liposomes did not alter the nonspecific uptake of QDs.Taken together, our data suggest that small-molecule fluorophores, not QDs, are suitable to detect the expression and downregulation of IGF1R in vivo.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT

Background: The type I insulin-like growth factor receptor (IGF1R) is a transmembrane tyrosine kinase involved in cancer proliferation, survival, and metastasis.

Methods: In this study, we used two different fluorescent technologies (small-molecule fluorophores and quantum dot (QD) nanoparticles) to detect receptor expression and its downregulation by antibodies in vivo.

Results: After conjugation with AVE-1642, a humanised anti-IGF1R monoclonal antibody, both QDs (705 nm) or Alexa 680 (small-molecule fluorophore) detected expression and downregulation of IGF1R in vitro. To examine their utility in vivo, either AVE-1642 conjugates were intravenously delivered to mice bearing xenograft tumours of mouse embryo fibroblasts expressing human IGF1R or MCF-7 human breast cancer cells. Quantum dot fluorescence was mainly localised to the reticuloendothelial system in several organs and engulfed by macrophages, with only very small amount of QDs detected in the xenograft tumours. Depletion of macrophages by clodronate liposomes did not alter the nonspecific uptake of QDs. In contrast, AVE-1642-conjugated Alexa 680 solely targeted to xenograft tumour and was able to detect IGF1R downregulation, with little nonspecific targeting to other tissues or organs in mice.

Conclusion: Taken together, our data suggest that small-molecule fluorophores, not QDs, are suitable to detect the expression and downregulation of IGF1R in vivo.

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Related in: MedlinePlus

Both AVE-1642-conjugated QDs and Alexa 680 bound to cells that express IGF1R. R cells, R-/IGF1R cells, or MCF-7 cells were trypsinised, resuspended in FACS buffer, and incubated with pure QDs, AVE-1642-conjugated QDs (AVE-QD 705) (A), AVE-1642-conjugated Alexa 680 (AVE-Alexa 680) (B), or both types of conjugates, including an anti-CD20 Alexa 680 conjugate (C) for 1 h. In addition, R-/IGF1R cells were pretreated with AVE-1642 (AVE) antibody for 24 h, and then incubated with AVE-QDs or AVE-Alexa 680. The fluorescence of bound QDs, or Alexa 680, was analysed by flow cytometry. The horizontal axis of the diagram represents the fluorescent intensity, and the vertical axis shows the percentage of maximum cell number.
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fig2: Both AVE-1642-conjugated QDs and Alexa 680 bound to cells that express IGF1R. R cells, R-/IGF1R cells, or MCF-7 cells were trypsinised, resuspended in FACS buffer, and incubated with pure QDs, AVE-1642-conjugated QDs (AVE-QD 705) (A), AVE-1642-conjugated Alexa 680 (AVE-Alexa 680) (B), or both types of conjugates, including an anti-CD20 Alexa 680 conjugate (C) for 1 h. In addition, R-/IGF1R cells were pretreated with AVE-1642 (AVE) antibody for 24 h, and then incubated with AVE-QDs or AVE-Alexa 680. The fluorescence of bound QDs, or Alexa 680, was analysed by flow cytometry. The horizontal axis of the diagram represents the fluorescent intensity, and the vertical axis shows the percentage of maximum cell number.

Mentions: Pure unconjugated QDs, or AVE-1642-conjugated QDs, were incubated with R cells, a mouse embryo fibroblast cell line that has genetic deletion of the IGF1R gene. After washing with FACS buffer, bound cell fluorescence was analysed by flow cytometry. No specific fluorescence was detected on cell surface. When R-/IGF1R cells, a cell line stably transfected with a human igf1r cDNA, were incubated with pure QDs or AVE-1642 QDs, only AVE-1642 QDs showed bound fluorescence. Pure QDs failed to bind to cell surface. In addition, if R-/IGF1R cells were pretreated with AVE-1642 antibody to downregulate IGF1R level, AVE-1642 QDs no longer bound to cell surface with a diminished IGF1R level (Figure 2A). Similar results were obtained with the AVE-1642-conjugated Alexa 680 (Figure 2B).


Fluorescent tumour imaging of type I IGF receptor in vivo: comparison of antibody-conjugated quantum dots and small-molecule fluorophore.

Zhang H, Zeng X, Li Q, Gaillard-Kelly M, Wagner CR, Yee D - Br. J. Cancer (2009)

Both AVE-1642-conjugated QDs and Alexa 680 bound to cells that express IGF1R. R cells, R-/IGF1R cells, or MCF-7 cells were trypsinised, resuspended in FACS buffer, and incubated with pure QDs, AVE-1642-conjugated QDs (AVE-QD 705) (A), AVE-1642-conjugated Alexa 680 (AVE-Alexa 680) (B), or both types of conjugates, including an anti-CD20 Alexa 680 conjugate (C) for 1 h. In addition, R-/IGF1R cells were pretreated with AVE-1642 (AVE) antibody for 24 h, and then incubated with AVE-QDs or AVE-Alexa 680. The fluorescence of bound QDs, or Alexa 680, was analysed by flow cytometry. The horizontal axis of the diagram represents the fluorescent intensity, and the vertical axis shows the percentage of maximum cell number.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713715&req=5

fig2: Both AVE-1642-conjugated QDs and Alexa 680 bound to cells that express IGF1R. R cells, R-/IGF1R cells, or MCF-7 cells were trypsinised, resuspended in FACS buffer, and incubated with pure QDs, AVE-1642-conjugated QDs (AVE-QD 705) (A), AVE-1642-conjugated Alexa 680 (AVE-Alexa 680) (B), or both types of conjugates, including an anti-CD20 Alexa 680 conjugate (C) for 1 h. In addition, R-/IGF1R cells were pretreated with AVE-1642 (AVE) antibody for 24 h, and then incubated with AVE-QDs or AVE-Alexa 680. The fluorescence of bound QDs, or Alexa 680, was analysed by flow cytometry. The horizontal axis of the diagram represents the fluorescent intensity, and the vertical axis shows the percentage of maximum cell number.
Mentions: Pure unconjugated QDs, or AVE-1642-conjugated QDs, were incubated with R cells, a mouse embryo fibroblast cell line that has genetic deletion of the IGF1R gene. After washing with FACS buffer, bound cell fluorescence was analysed by flow cytometry. No specific fluorescence was detected on cell surface. When R-/IGF1R cells, a cell line stably transfected with a human igf1r cDNA, were incubated with pure QDs or AVE-1642 QDs, only AVE-1642 QDs showed bound fluorescence. Pure QDs failed to bind to cell surface. In addition, if R-/IGF1R cells were pretreated with AVE-1642 antibody to downregulate IGF1R level, AVE-1642 QDs no longer bound to cell surface with a diminished IGF1R level (Figure 2A). Similar results were obtained with the AVE-1642-conjugated Alexa 680 (Figure 2B).

Bottom Line: After conjugation with AVE-1642, a humanised anti-IGF1R monoclonal antibody, both QDs (705 nm) or Alexa 680 (small-molecule fluorophore) detected expression and downregulation of IGF1R in vitro.Depletion of macrophages by clodronate liposomes did not alter the nonspecific uptake of QDs.Taken together, our data suggest that small-molecule fluorophores, not QDs, are suitable to detect the expression and downregulation of IGF1R in vivo.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT

Background: The type I insulin-like growth factor receptor (IGF1R) is a transmembrane tyrosine kinase involved in cancer proliferation, survival, and metastasis.

Methods: In this study, we used two different fluorescent technologies (small-molecule fluorophores and quantum dot (QD) nanoparticles) to detect receptor expression and its downregulation by antibodies in vivo.

Results: After conjugation with AVE-1642, a humanised anti-IGF1R monoclonal antibody, both QDs (705 nm) or Alexa 680 (small-molecule fluorophore) detected expression and downregulation of IGF1R in vitro. To examine their utility in vivo, either AVE-1642 conjugates were intravenously delivered to mice bearing xenograft tumours of mouse embryo fibroblasts expressing human IGF1R or MCF-7 human breast cancer cells. Quantum dot fluorescence was mainly localised to the reticuloendothelial system in several organs and engulfed by macrophages, with only very small amount of QDs detected in the xenograft tumours. Depletion of macrophages by clodronate liposomes did not alter the nonspecific uptake of QDs. In contrast, AVE-1642-conjugated Alexa 680 solely targeted to xenograft tumour and was able to detect IGF1R downregulation, with little nonspecific targeting to other tissues or organs in mice.

Conclusion: Taken together, our data suggest that small-molecule fluorophores, not QDs, are suitable to detect the expression and downregulation of IGF1R in vivo.

Show MeSH
Related in: MedlinePlus