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In vivo imaging of colorectal cancer growth and metastasis by targeting MACC1 with shRNA in xenografted mice

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Abstract

We previously identified the gene metastasis-associated in colon cancer-1 (MACC1) and demonstrated its important role for metastasis prediction in colorectal cancer. MACC1 induces cell motility and proliferation in vitro as well as metastasis in several mouse models. Here we report non-invasive real time imaging of inhibition of colorectal tumor progression and metastasis in xenografted mice by MACC1 shRNA. First, we demonstrated reduction of tumors and liver metastases by endpoint imaging of mice transplanted with MACC1 endogenously high expressing colorectal cancer cells and treated with shRNAs acting on MACC1 or Met. Next, we generated a novel bicistronic IRES vector simultaneously expressing the reporter gene firefly luciferase and MACC1 to ensure a direct correlation of bioluminescence signal with MACC1 expression. We transfected MACC1 endogenously low expressing colorectal cancer cells with this luciferase-IRES-MACC1 construct, transplanted them intrasplenically, and monitored MACC1 induced tumor growth and metastasis by in vivo imaging over time. Transfection of an IRES construct harboring the firefly luciferase reporter gene together with MACC1 lacking the SH3-domain reduced tumor growth and metastasis. Finally, we counteracted the luciferase-IRES-MACC1 induced effects by shRNA targeting MACC1 and monitored reduced tumor growth and metastasis by in vivo imaging over weeks. In summary, the new bicistronic luciferase-IRES-MACC1 construct is suitable for in vivo imaging of tumor progression and metastasis, and moreover, for imaging of therapy response such as treatment with MACC1 shRNA. Thereby, we provide proof-of-concept for employment of this MACC1-based in vivo model for evaluating therapeutic intervention strategies aiming at inhibition of tumor growth and metastasis.

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Abbreviations

CRC:

Colorectal cancer

DMSO:

Dimethylsulfoxide

FBS:

Fetal bovine serum

HGF:

Hepatocyte growth factor

IRES:

Internal ribosomal entry site

Luc:

Luciferase

MACC1:

Metastasis-associated in colon cancer-1

MACC1ΔSH3:

MACC1 with a deleted SH3-domain

MAPK:

Mitogen-activated protein kinase

PBS:

Phosphate buffered saline

PCR:

Polymerase chain reaction

PXXP:

Proline-rich motif

RT-PCR:

Reverse-transcriptase polymerase chain reaction

SCID:

Severe combined immune deficient

SH3:

Src-homology 3

shRNA:

Small hairpin RNA

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Acknowledgments

We are very grateful to Pia Hermann and Jutta Aumann for their technical assistance and to Mathias Dahlmann, Felicitas Schmid, Clara Lemos, Anne Enders and Patrick Tauscher for their methodological and scientific advice.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Experimental and Clinical Research Center, Charité University Medicine Berlin, at the Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125, Berlin, Germany

    Andreas Pichorner, Inken Kelch, Franziska Arlt, Wolfgang Walther & Ulrike Stein

  2. Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125, Berlin, Germany

    Ulrike Sack, Dennis Kobelt & Janice Smith

  3. Charité Comprehensive Cancer Center, Invalidenstraße 80, 10117, Berlin, Germany

    Peter M. Schlag

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  1. Andreas Pichorner
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  2. Ulrike Sack
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  3. Dennis Kobelt
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  4. Inken Kelch
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  5. Franziska Arlt
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  6. Janice Smith
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  7. Wolfgang Walther
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  8. Peter M. Schlag
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  9. Ulrike Stein
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Correspondence to Ulrike Stein.

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Pichorner, A., Sack, U., Kobelt, D. et al. In vivo imaging of colorectal cancer growth and metastasis by targeting MACC1 with shRNA in xenografted mice. Clin Exp Metastasis 29, 573–583 (2012). https://doi.org/10.1007/s10585-012-9472-6

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  • DOI: https://doi.org/10.1007/s10585-012-9472-6

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