Elsevier

Experimental Cell Research

Volume 314, Issue 17, 15 October 2008, Pages 3107-3117
Experimental Cell Research

Research Article
The isolation of novel mesenchymal stromal cell chemotactic factors from the conditioned medium of tumor cells

https://doi.org/10.1016/j.yexcr.2008.07.028Get rights and content

Abstract

Bone marrow-derived mesenchymal stromal cells (MSCs) localize to solid tumors. Defining the signaling mechanisms that regulate this process is important in understanding the role of MSCs in tumor growth. Using a combination of chromatography and electrospray tandem mass spectrometry we have identified novel soluble signaling molecules that induce MSC chemotaxis present in conditioned medium of the breast carcinoma cell line MDA-MB231. Previous work has employed survey strategies using ELISA assay to identify known chemokines that promote MSC chemotaxis. While these studies provide valuable insights into the intercellular signals that impact MSC behavior, many less well-described, but potentially important soluble signaling molecules could be overlooked using these methods. Through the less directed method of column chromatography we have identified novel candidate MSC chemotactic peptides. Two proteins, cyclophilin B and hepatoma-derived growth factor were then further characterized and shown to promote MSC chemotaxis.

Introduction

Multipotent mesenchymal stromal cells (MSCs) are bone marrow-derived plastic-adherent cells that were initially described by Friedenstein and colleagues [1], [2]. They have a fibroblast-like phenotype [1], [2] and can be differentiated along osteoblastic, chondrocytic, adipogenic, and myofibroblastic lineages [3], [4]. MSCs within the bone marrow support hematopoiesis and the recent identification of MSCs in the peripheral blood [5], [6], [7], [8] as well as tissues such as fat [9] and placenta [10] support the hypothesis that MSCs have important functions outside of the bone marrow. Systemically delivered MSCs localize to areas of active inflammation including sites of bone fracture [11], cutaneous incisional wounds [12], myocardial infarction [13] and solid tumors [14], [15], [16] indicating that these cells are involved in tissue repair and growth.

While the propensity of MSCs to migrate to tumors and areas of tissue damage has been well-documented, the molecular signals guiding this movement are not completely defined. MSCs are known to respond to a number of chemotactic factors including stromal-derived factor-1 (SDF-1) [17], [18], vascular endothelial growth factor A (VEGF-A) [18], [19], platelet derived growth factor (PDGF) [20], monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1-alpha (MIP-1α), and interleukin 8 (IL-8) [21]. Tumor cells are a potential source of MSC chemotactic factors and we have previously shown that tumor cell line conditioned medium promotes MSC chemotaxis [22].

Proteoglycans are found on cell surfaces and within the extracellular matrix and have been implicated in several aspects of cell-to-cell communication including the interaction of chemokines with cell surface receptors, protection of signaling peptides from degradation, and facilitating the formation of chemokine gradients [23], [24], [25]. We therefore hypothesized that factors that induce MSC chemotaxis may bind proteoglycans and used heparin sulfate column chromatography as the basis for enrichment of pro-chemotactic molecules from tumor-conditioned medium. Peptides within the fraction of conditioned medium with chemotactic activity were identified using electrospray tandem mass spectrometry protein sequencing. Of these, cyclophilin B and hepatoma-derived growth factor were further investigated and found to promote MSC chemotaxis.

Using proteomic techniques we have identified candidate chemotactic molecules for MSCs that have not been previously recognized using molecular genetic or ELISA-based identification strategies.

Section snippets

Antibodies

The monoclonal antibody against CypB (4E11G1) was obtained from Invitrogen (Carlsbad, CA). The anti-CD147 antibody (HIM6) was purchased from BD Biosciences (Franklin Lakes, NJ). The HDGF-blocking antibody (C-14) and anti-osteocalcin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Isolation and culture of Mesenchymal Stem Cells

Rat MSCs were isolated as previously described [22]. Briefly, rats were euthanized by CO2 inhalation and the bilateral femora and tibias were dissected under aseptic conditions and washed in

Purification and identification of pro-migratory factors from tumor-conditioned medium

Using a Boyden chamber migration assay we developed a two-step purification procedure for the enrichment of heparin-binding molecules that induce MSC migration from tumor-conditioned medium from the human breast cancer cell line MDA-MB231 (Fig. 1A). Rat MSCs were used in the migration assay throughout the purification procedure. The tumor-conditioned medium was first loaded onto a heparin column, protein fractions were eluted using a step gradient of 0.2–0.8 M NaCl and fractions were assayed

Discussion

Bone marrow-derived MSCs localize to solid tumors and may play an important role within the tumor stroma. In an effort to understand the molecular signals involved in MSC migration, factors that promote MSC chemotaxis have been identified using molecular genetic approaches and ELISA analysis. While important information has been obtained using these methods, less well-described chemotactic stimuli may be overlooked. We have used heparin binding, a specific biochemical property common to

Acknowledgments

SYL was supported by NIH grant T32 CA 108455. This work was supported by a collaborative research grant from the Cancer Institute of New Jersey and research grants from The New Jersey Commission on Science and Technology (HESC-06-04-00) and The New Jersey Commission on Cancer Research (05-2406-CCR-EO). Additional support was provided by the AHEPA Cancer Research Foundation.

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