Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Oct;58(10):1577-86.
doi: 10.1007/s00262-009-0667-x. Epub 2009 Feb 24.

Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor beta

Taku Nagai  1 , Masashi Tanaka, Yasuhiro Tsuneyoshi, Baohui Xu, Sara A Michie, Kazuhisa Hasui, Hirofumi Hirano, Kazunori Arita, Takami Matsuyama
Affiliations

Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor beta

Taku Nagai et al. Cancer Immunol Immunother. 2009 Oct.

Abstract

Tumor-associated macrophages (TAMs) are frequently found in glioblastomas and a high degree of macrophage infiltration is associated with a poor prognosis for glioblastoma patients. However, it is unclear whether TAMs in glioblastomas promote tumor growth. In this study, we found that folate receptor beta (FR beta) was expressed on macrophages in human glioblastomas and a rat C6 glioma implanted subcutaneously in nude mice. To target FR beta-expressing TAMs, we produced a recombinant immunotoxin consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FR beta monoclonal antibody and Pseudomonas exotoxin A. Injection of the immunotoxin into C6 glioma xenografts in nude mice significantly depleted TAMs and reduced tumor growth. The immunotoxin targeting FR beta-expressing macrophages will provide a therapeutic tool for human glioblastomas.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Distribution of FRβ-expressing macrophages in human glioblastoma. A human glioblastoma was stained with, A mAb against CD68 (left) or FRβ (middle) (original magnification is 100×), or with anti-CD68 mAb (blue) and anti-FRβ mAb (red) (right) (original magnification is 400×) and B mAb against CD163 (left) or FRβ (middle) (original magnification is 100×), or with anti-CD163 mAb (blue) and anti-FRβ mAb (red) (right) (original magnification is 400×) (color in online)
Fig. 2
Fig. 2
Reactivity of anti-mouse FRβ mAb. A FRβ-transfected B300-19 cells (left), non-transfected B300-19 cells (middle), and FRα-expressing M109 cells (right) were stained with an anti-mouse FRβ (CL5) mAb or an isotype control (IgG2a) mAb and were analyzed by flow cytometry. White figures are stained with the control mAb and black figures are stained with the anti-FRβ. The same data were obtained using anti-mouse FRβ (CL10) mAb. B Thioglycollate-elicited peritoneal macrophages were stained with APC-conjugated isotype control mAb (left) or APC-anti-FRβ (CL5) mAb (right) and FITC-conjugated isotype control mAb (left) or FITC-anti-F4/80 mAb (right) and analyzed by flow cytometry. The same data were obtained using anti-mouse FRβ (CL10) mAb. C The lysates from FRβ-transfected B300-19 cells (lanes a and c) or non-transfected B300-19 cells (lanes b and d) were run on 12% SDS-PAGE (non-reducing conditions), transferred to PVDF and the blots were reacted with anti- FRβ (CL5) (lanes a and b) or control mAbs (lanes c and d). Molecular weight markers are shown at the right. The same data were obtained using anti-mouse FRβ (CL10) mAb
Fig. 3
Fig. 3
Effects of a recombinant immunotoxin to FRβ on thioglycollate-elicited peritoneal macrophages. A The recombinant immunotoxin, dsFv anti- FRβ-PE38 was run on 12% SDS-PAGE under non-reducing (-SH) and reducing conditions (+SH). Molecular weight markers are shown at the right. B Thioglycollate-elicited peritoneal macrophages were cultured in the presence of dsFv anti-FRβ-PE38 or VH-PE38 at indicated concentrations for 48, 72 and 96 h (filled circle). The percentages of apoptotic cells (sub G0/G1 cells) were measured by propidium iodide staining and flow cytometry. Induced apoptosis (%) was determined by subtracting the percentage of apoptotic cells induced by VH-PE38 from that induced by dsFv anti-FRβ-PE38 in each sample. Values (%) are the mean ± SEM of three separate experiments. C, D Thioglycollate-elicited peritoneal macrophages were cultured in the presence of dsFv anti-FRβ-PE38 (filled circle), VH-PE38 (open circle) or medium only for 24 h and stimulated with IFN-γ and LPS for another 24 h. NO and VEGF in the supernatants were measured as described in “Materials and methods”. NO and VEGF production in the presence of dsFv anti-FRβ-PE38 or VH-PE38 is given as the percentage of that in the medium-only control (The concentrations of NO and VEGF were 39.6 ± 1.5 μM and 83.5 ± 1.9 pg/ml, respectively). Values (%) are the mean ± SEM of three separate experiments. * P < 0.05 compared to VH-PE38 group
Fig. 4
Fig. 4
Effect of dsFv anti-FRβ-PE38 on rat C6 glioma growth Rat C6 gliomas were injected intratumorly with dsFv anti-FRβ-PE38 at 0.5 μg (filled triangle) and 2.0 μg (white square) per tumor, VH-PE38 (white circle) at 2.0 μg per tumor or equal volume of saline (filled circle) on days 3, 5, 7, 9, 11, and 13. The tumor volume was measured as described in “Materials and methods”. Values are the mean ± SEM of the tumor volume of six tumors per group. * P < 0.05 compared to VH-PE38 group
Fig. 5
Fig. 5
Immunohistochemical analysis of rat C6 gliomas treated with a recombinant immunotoxin to FRβ. Frozen sections of tumors in each group were stained with mAb against F4/80, FRβ or CD31. A Representative image of rat C6 glioma tissues stained with mAb against F4/80 (left panels), FRβ (CL5) (middle panels), and CD31 (right panels) in dsFv anti-FRβ-PE38-treated (top panels) and VH-PE38-treated (bottom panels) groups. B F4/80-expressing cells (left), FRβ-expressing cells (middle) or CD31-positive vascular area (right) were measured as described in “Materials and methods”. Values are the mean ± SEM of F4/80-expressing cells, FRβ-expressing cells, or CD31-positive vascular area of six tumors in each group. * P < 0.05 compared to VH-PE38 group
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bacman D, Merkel S, Croner R, Papadopoulos T, Brueckl W, Dimmler A. TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study. BMC Cancer. 2007;7:156. doi: 10.1186/1471-2407-7-156. - DOI - PMC - PubMed
    1. Bolat F, Kayaselcuk F, Nursal TZ, Yagmurdur MC, Bal N, Demirhan B. Microvessel density, VEGF expression, and tumor-associated macrophages in breast tumors: correlations with prognostic parameters. J Exp Clin Cancer Res. 2006;25:365–372. - PubMed
    1. Chen JJ, Lin YC, Yao PL, Yuan A, Chen HY, Shun CT, Tsai MF, Chen CH, Yang PC. Tumor-associated macrophages: the double-edged sword in cancer progression. J Clin Oncol. 2005;23:953–964. doi: 10.1200/JCO.2005.12.172. - DOI - PubMed
    1. Deininger MH, Meyermann R, Schluesener HJ. Expression and release of CD14 in astrocytic brain tumors. Acta Neuropathol. 2003;106:271–277. doi: 10.1007/s00401-003-0727-9. - DOI - PubMed
    1. Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol. 2002;196:254–265. doi: 10.1002/path.1027. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources