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Development and characterization of a reliable mouse model of colorectal cancer metastasis to the liver

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Abstract

Colorectal cancer (CRC) is the third most frequent cancer and the third leading cause of cancer deaths in the United States (American Cancer Society, Cancer facts and figures 2012, 20121). The major cause of death is metastasis and frequently, the target organ is the liver. Successful metastasis depends on acquired properties in cancer cells that promote invasion and migration, and on multiple interactions between tumors and host-derived cells in the microenvironment. These processes, however, occur asymptomatically, thus, metastasis remains poorly understood and often diagnosed only at the final stage. To facilitate the elucidation of the mechanisms underlying these processes and to identify the molecular regulators, particularly at the early stages, we developed a mouse model of hepatic metastasis of CRC by cecal implantation of a mouse adenocarcinoma cell line in an immune competent host that reliably recapitulates all steps of tumor growth and metastasis within a defined period. By in vivo selection, we isolated cells of varying metastatic potential. The most highly metastatic CT26-FL3 cells produced liver metastasis as early as 10 days after implantation in 90 % of host mice. These cells expressed elevated levels of genes whose products promote invasion, migration, and mobilization of bone marrow derived cells (BMDCs). Mice bearing tumors from CT26-FL3 had elevated serum levels of OPN, MMP9, S100A8, S100A9, SAA3, and VEGFA that promote invasion and BMDC mobilization, and showed enhanced BMDC recruitment to the liver where they established a pre-metastatic niche. This model provides an important platform to characterize metastatic cells and elucidate tumor–host interactions and mechanisms that drive liver metastasis of CRC.

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Abbreviations

CRC:

Colorectal cancer

BMDC:

Bone marrow derived cells

PMN:

Pre-metastatic niche

HSC:

Hematopoietic stem cells

BM:

Bone marrow

BMT:

Bone marrow transplantation

PCNA:

Proliferating Cell Nuclear Antigen

MMP:

Matrix metalloproteinase

VEGF:

Vascular endothelial growth factor

VEGF-R1:

VEGF receptor 1

LOX:

Lysyl oxidase

CCND1:

Cyclin D1

OPN:

Osteopontin

SAA3:

Serum amyloid A3

HRP:

Horseradish peroxidase

HGF:

Hepatocyte growth factor

IL-6:

Interleukin 6

TNF-α:

Tumor necrosis factor alpha

IFN-γ:

Interferon gamma

CSF:

Colony stimulating factor

LLC:

Lewis lung carcinoma

eGFP:

Enhanced green fluorescent protein

RFP:

Red fluorescent protein

MDSC:

Myeloid derived suppressor cells

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Acknowledgments

The authors would like to Ms. Anna McNeal Harper and Dr. Udai Singh of the Biotechnology Core, USC Scholl of Medicine, for technical support in confocal microscopy and flow cytometry, respectively, and Ms. Julia Long, Palmetto Richland Radiology and Oncology for assistance with mouse irradiation. This study was supported by NIH Grants P20 RR17698 as a Target Principal Investigator of the USC Center for Colon Cancer Research and R01CA154731-1A1.

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Authors and Affiliations

  1. Department of Biological Sciences, University of South Carolina, Columbia, SC, 29208, USA

    Yu Zhang, Celestia Davis, Cory Janney & Maria Marjorette O. Peña

  2. NOAA/Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC, 29412, USA

    James Ryan

  3. Center for Colon Cancer Research, University of South Carolina, Columbia, SC, 29208, USA

    Maria Marjorette O. Peña

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  1. Yu Zhang
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  2. Celestia Davis
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Correspondence to Maria Marjorette O. Peña.

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10585_2013_9591_MOESM1_ESM.tif

Supplemental Fig. 1 Migration of eGFP positive BMDCs into the liver prior to the arrival to tumor cells mCherry-RFP positive tumor cells at 3 weeks post cecal implantation. Liver sections were analyzed for green fluorescence (eGFP), red fluorescence (mCherry), Phalloidin (actin) and DAPI (blue). Cells implanted with CT26-FL3 showed the presence of BMDCs and the absence of tumor cells (upper panel) while both green and red fluorescence were absent in control Sham injected animals (lower panel)

10585_2013_9591_MOESM2_ESM.tif

Supplemental Fig. 2. Co-localization of markers associated with pre-metastatic niche formation with BMDCs infiltrating the liver prior to the arrival of metastasizing tumor cells. Balbc/ByJ mice were transplanted with BM from transgenic mice expressing eGFP prior to implantation of CT26-FL3 cells into the cecum. Liver sections were taken 2.5 weeks post cecal implantation. The sections were stained with antibodies against S100A8 (top panel), S100A9 (second panel), LOX (third panel), and VEGF-R1 (bottom panel), then examined for the presence of BMDCs (Green), and counterstained with DAPI (Blue). Red = Positive staining for protein specific antibodies. Images were the merged to determine co-localization of the protein markers with the eGFP positive BMDCs

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Zhang, Y., Davis, C., Ryan, J. et al. Development and characterization of a reliable mouse model of colorectal cancer metastasis to the liver. Clin Exp Metastasis 30, 903–918 (2013). https://doi.org/10.1007/s10585-013-9591-8

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  • DOI: https://doi.org/10.1007/s10585-013-9591-8

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