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Cancer associated fibroblasts stimulated by transforming growth factor beta1 (TGF-β1) increase invasion rate of tumor cells: a population study

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Abstract

Cancer associated fibroblasts (CAFs) are believed to promote tumor growth and progression. Our objective was to measure the effect of TGF-β1 on fibroblasts isolated from invasive breast cancer patients. Fibroblasts were isolated from tissue obtained at surgery from patients with invasive breast cancer (CAF; n = 28) or normal reduction mammoplasty patients (normal; n = 10). Myofibroblast activation was measured by counting cells immunostained for smooth muscle alpha actin (ACTA2) in cultures ± TGF-β1. Conditioned media (CM) was collected for invasion assays and RNA was isolated from cultures incubated in media ± TGF-β1 for 24 h. Q-PCR was used to measure expression of cyclin D1, fibronectin, laminin, collagen I, urokinase, stromelysin-1, and ACTA2 genes. Invasion rate was measured in chambers plated with MDA-MB-231 cells and exposed to CM in the bottom chamber; the number of cells that invaded into the bottom chamber was counted. Wilcox Rank Sum tests were used to evaluate differences in CAFs and normal fibroblasts and the effect of TGF-β1. There was no difference in percent myofibroblasts or invasion rate between normal and CAF cultures. However, TGF-β1 significantly increased the percent of myofibroblasts (P < 0.01) and invasion rate (P = 0.02) in CAF cultures. Stromelysin-1 expression was significantly higher in normal versus CAF cultures (P < 0.01). TGF-β1 significantly increased ACTA2 expression in both normal and CAF cultures (P < 0.01). Expression of fibronectin and laminin was significantly increased by TGF-β in CAF cultures (P < 0.01). CAFs were measurably different from normal fibroblasts in response to TGF-β1, suggesting that TGF-β stimulates changes in CAFs that foster tumor invasion.

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Abbreviations

CAF:

Cancer associated fibroblast

TGF-β:

Transforming growth factor-beta

ECM:

Extracellular matrix

FBS:

Fetal bovive serum

BM:

Basal media

CM:

Conditioned media

ACTA2:

Smooth muscle alpha actin

CCND1:

Cyclin D1

FN1:

Fibronectin

LAMA1:

Laminin

COL1A1:

Collagen I

MMP3:

Stromelysin-1

PLAU:

Urokinase

RQ:

Relative expression

CT:

Cycle threshold

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Acknowledgments

This work was supported by a grant from The Breast Cancer Research Foundation, New York, NY Real-time quantitative PCR was performed in the Vermont Cancer Center DNA Analysis Facility and was supported in part by grant P30CA22435 from the NCI.

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Authors and Affiliations

  1. Department of Animal Science, Michigan State University, B290 Anthony Hall, East Lansing, MI, 48824, USA

    Theresa M. Casey & Karen Plaut

  2. Department of Hematology and Oncology, University of Vermont, Burlington, VT, 05405, USA

    Jonathan Eneman, Abigail Crocker & Hyman Muss

  3. Department of Animal Science, University of Vermont, Burlington, VT, 05405, USA

    Jeffrey White

  4. Department of Pathology, University of Vermont, Burlington, VT, 05405, USA

    Joseph Tessitore & Donald Weaver

  5. Department of Surgery, University of Vermont, Burlington, VT, 05405, USA

    Mary Stanley & Seth Harlow

  6. Department of Medical Biostatistics, University of Vermont, Burlington, VT, 05405, USA

    Janice Y. Bunn

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  1. Theresa M. Casey
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Correspondence to Theresa M. Casey.

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Casey, T.M., Eneman, J., Crocker, A. et al. Cancer associated fibroblasts stimulated by transforming growth factor beta1 (TGF-β1) increase invasion rate of tumor cells: a population study. Breast Cancer Res Treat 110, 39–49 (2008). https://doi.org/10.1007/s10549-007-9684-7

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