Secretion of MCP-1 and other paracrine factors in a novel tumor-bone coculture model

Abstract

BackgroundThe bone-tumor microenvironment encompasses unique interactions between the normal cells of the bone and marrow cavity and the malignant cells from a primary or metastasized cancer. A multitude of paracrine factors within this microenvironment such as the growth factor, TGF-β, and the chemokine, MCP-1, are secreted by many of these cell types. These factors can act in concert to modulate normal and malignant cell proliferation, malignant cell migration and invasion and, often, mediate bone cancer pain. Although many valuable in vitro and in vivo models exist, identifying the relevant paracrine factors and deciphering their interactions is still a challenge. The aim of our study is to test an ex vivo coculture model that will allow monitoring of the expression, release and regulation of paracrine factors during interactions of an intact femur explant and tumor cells.MethodsIntact or marrow-depleted neonatal mouse femurs and select murine and human sarcoma or carcinoma cell lines were incubated singly or in coculture in specialized well plates. Viability of the bone and cells was determined by immunohistochemical stains, microscopy and marrow cytopreps. Secretion and mRNA expression of paracrine factors was quantitated by ELISA and real-time RT-PCR.ResultsCompartments of the bone were optimally viable for up to 48 h in culture and tumor cells for up to 4 days. Bone was the major contributor of TGF-β and MMP2 whereas both bone and sarcoma cells secreted the chemokine MCP-1 in cocultures. Synergistic interaction between the femur and sarcoma resulted in enhanced MCP-1 secretion and expression in cocultures and was dependent on the presence of the hematopoietic component of the bone as well as other bone cells. In contrast, coculturing with breast carcinoma cells resulted in reduction of TGF-β and MCP-1 secretion from the bone.ConclusionThese studies illustrate the feasibility of this model to examine paracrine interactions between intact bone and tumor cells. Further study of unique regulation of MCP-1 secretion and signaling between these cell types in different types of cancer will be possible using this simulated microenvironment.

Monitoring Dynamic Interactions Between Breast Cancer Cells and Human Bone Tissue in a Co-culture Model

“…In keeping with other reports, we observed a decline in marrow viability after 48 hours as revealed by trypan blue exclusion measurement and appearance in histologic section [31]. An important next step will involve the systematic optimization of conditions for long-term culture.…”

“…Schiller et al developed a model in which whole neonatal mouse femurs were co-cultured with a variety of mouse and human cancer cell lines, demonstrating bone tissue viability and illustrating synergistic paracrine interactions between intact bone and tumor cells with enzyme-linked immunosorbent assay (ELISA) [31]. Curtin et al developed a roller tube model system to co-culture mouse calvarial bones with breast and prostate cancer cell lines to demonstrate cancer cell colonization of the endosteal bone surfaces and subsequent promotion of osteoclast activity resulting in bone resorption [32].…”

Cancer-induced bone pain: Mechanisms and models

“…Development of co- cultures with femur and cancer cells, up-regulate MCP-1 and TGFβ expression and secretion in sarcoma, but down-regulates MCP-1 and TGFβ expression and secretion in breast cancer cultures [85]. However, recent studies measuring cytokines and chemokines in vivo in a murine model of breast cancer demonstrated a significant increase in MCP-1, MIP-1A, IL-6 and TNFα [61].…”

MCP1 Directs Trafficking of Hematopoietic Stem Cell-Derived Fibroblast Precursors in Solid Tumor

“…CFPs were placed into transwell culture plates with control media, tumor-CM, or tumor-CM with the addition of MCP1 neutralizing antibody. Tumor-CM was harvested from the LLC, EMT-6 (murine mammary carcinoma) and K7M2 (murine osteosarcoma) tumor cell lines, all of which have been shown to produce MCP1 (data above and references 43,44 ). Analysis showed that exposure to conditioned media from each tumor line resulted in preferential migration of CFPs when compared with response of CFPs to control media ( Figure 11G, P Յ 0.05).…”