Microdialysis combined with proteomics for protein identification in breast tumor microenvironment in vivo

Abstract

Tumor microenvironment constitutes a reservoir for proteins released from tumor cells and the host, which can contribute significantly to tumor growth and invasion. This study aims to apply a method of combining in vivo microdialysis and proteomics to identify proteins in mammary tumor interstitial fluids, a major component of tumor microenvironment. In vivo microdialysis was performed in polyomavirus middle T antigen (PyVmT) transgenic mouse mammary tumors and age-matched control wild-type mammary glands. Over four hundred proteins were identified from the microdialysis perfusates, using the Multidimensional Protein Identification Technology. Osteopontin (OPN) is one of the proteins overexpressed in breast tumor perfusates, as confirmed with immunoassays. OPN was also found to be present in tumor-associated stroma in both PyVmT and human breast tumors, using immunohistochemistry. Specifically, fibroblasts were further shown to express OPN at both mRNA and protein levels. In vitro assays showed that OPN can stimulate PyVmT breast carcinoma cell proliferation and migration. Finally, the expression of OPN was significantly higher in the peripheral blood of mice bearing breast tumors, compared to wild-type mice. Overall, microdialysis combined with proteomics is a unique technique for identifying proteins in a tumor microenvironment in vivo. Mammary fibroblasts can secrete OPN, and its overexpression in mammary tumor microenvironment may contribute significantly to mammary tumor progression.

Electronic supplementary material: The online version of this article (doi:10.1007/s12307-010-0046-3) contains supplementary material, which is available to authorized users.

Keywords: Fibroblasts; Microdialysis; Osteopontin; Proteomics; Tumor microenvironment.

Fig. 1

The microdialysis probes embedded in tissue. a–b The microdialysis probes in the normal mammary fat pad and solid mammary tumor, respectively. c–d H &E stained sections demonstrate that microdialysis probes were adjacent to normal mammary gland adipose cells or mammary tumor cells, respectively

Fig. 2

Protein identification from microdialysis perfusates and related bioinformatic analyses. a the number of proteins identified from normal mammary gland and tumor microdialysis perfusates. b The distribution of the theoretical molecular weight of the identified proteins. c The distribution of the theoretical isoelectrical points of the identified proteins. d The potential cellular distribution of the identified proteins

Fig. 3

Confirmation of the differentially expressed proteins, clusterin, cystatin-C and osteopontin, using Luminex based immunoassay. Microdialysis perfusates obtained from mammary tumor and normal mammary glands were analyzed and significant differences in the protein expressions were observed (p < 0.005)

Fig. 4

Immunohistochemistry of OPN in wild-type mouse mammary gland, PyVmT breast tumor and human breast cancer. a OPN was mainly found in the lumen of the ducts in normal mammary gland but not in the adipose tissue. b OPN was found to be presented in both PyVmT carcinoma cells and the tumor associated stroma. c OPN was found in human breast cancer associated stroma

Fig. 5

Examination of OPN mRNA in mouse and human breast cancer and fibroblast cell lines. a A strong correlation was observed between mRNA- and protein-level expression of OPN in multiple primary mouse fibroblasts. b Both primary mouse PyVmT (N = 2) and fibroblasts cell lines (N = 2) express OPN mRNA. c OPN mRNA was expressed at higher levels in human breast fibroblast cell lines (N = 2) compared with carcinoma cell lines (N = 2)

Fig. 6

a OPN stimulates PyVmT carcinoma cells proliferation using WST-1 assay. b OPN stimulates PyVmT carcinoma cells migration using wound closure assay. c The sera OPN protein expression in PyVmT-tumor bearing mice (N = 5) is significantly higher than its expression in the sera from wild-type mice (N = 5)