External Beam Radiation Therapy Enhances Mesenchymal Stem Cell-Mediated Sodium-Iodide Symporter Gene Delivery.

The tumor-homing properties of mesenchymal stem cells (MSC) have led to their development as delivery vehicles for the targeted delivery of therapeutic genes such as the sodium-iodide symporter (NIS) to solid tumors. External beam radiation therapy may represent an ideal setting for the application of engineered MSC-based gene therapy, as tumor irradiation may enhance MSC recruitment into irradiated tumors through the increased production of select factors linked to MSC migration. In the present study, the irradiation of human liver cancer cells (HuH7; 1-10 Gy) showed a strong dose-dependent increase in steady-state mRNA levels of CXCL8, CXCL12, FGF2, PDGFB, TGFB1, THBS1, and VEGF (0-48 h), which was verified for most factors at the protein level (after 48 h). Radiation effects on directed MSC migration were tested in vitro using a live cell tracking migration assay and supernatants from control and irradiated HuH7 cells. A robust increase in mean forward migration index, mean center of mass, and mean directionality of MSCs toward supernatants was seen from irradiated as compared to non-irradiated tumor cells. Transferability of this effect to other tumor sources was demonstrated using the human breast adenocarcinoma cell line (MDA-MB-231), which showed a similar behavior to radiation as seen with HuH7 cells in quantitative polymerase chain reaction and migration assay. To evaluate this in a more physiologic in vivo setting, subcutaneously growing HuH7 xenograft tumors were irradiated with 0, 2, or 5 Gy followed by CMV-NIS-MSC application 24 h later. Tumoral iodide uptake was monitored using 123I-scintigraphy. The results showed increased tumor-specific dose-dependent accumulation of radioiodide in irradiated tumors. The results demonstrate that external beam radiation therapy enhances the migratory capacity of MSCs and may thus increase the therapeutic efficacy of MSC-mediated NIS radionuclide therapy.

Thyroid hormones and tetrac: new regulators of tumour stroma formation via integrin αvß3.

To improve our understanding of non-genomic, integrin αvß3-mediated thyroid hormone action in tumour stroma formation, we examined the effects of triiodo-l-thyronine (T3), l-thyroxine (T4) and integrin-specific inhibitor tetrac on differentiation, migration and invasion of mesenchymal stem cells (MSCs) that are an integral part of the tumour's fibrovascular network. Primary human bone marrow-derived MSCs were treated with T3 or T4 in the presence of hepatocellular carcinoma (HCC) cell-conditioned medium (CM), which resulted in stimulation of the expression of genes associated with cancer-associated fibroblast-like differentiation as determined by qPCR and ELISA. In addition, T3 and T4 increased migration of MSCs towards HCC cell-CM and invasion into the centre of three-dimensional HCC cell spheroids. All these effects were tetrac-dependent and therefore integrin αvß3-mediated. In a subcutaneous HCC xenograft model, MSCs showed significantly increased recruitment and invasion into tumours of hyperthyroid mice compared to euthyroid and, in particular, hypothyroid mice, while treatment with tetrac almost completely eliminated MSC recruitment. These studies significantly improve our understanding of the anti-tumour activity of tetrac, as well as the mechanisms that regulate MSC differentiation and recruitment in the context of tumour stroma formation, as an important prerequisite for the utilisation of MSCs as gene delivery vehicles.

Quantification of in vitro mesenchymal stem cell invasion into tumor spheroids using selective plane illumination microscopy.

Mesenchymal stem cell (MSC) homing and integration into tumors are under evaluation for clinical application. This approach requires the identification of conditions for optimal tumor invasion. We describe a tool for the in vitro comparison of parameters influencing invasion. Human MSC added to experimental tumor spheroids variably migrates toward the center of the structure. To determine MSC distribution inside the three-dimensional specimen, spatial analysis was performed using selective plane illumination microscopy. A standardized method to quantify and compare the invasion potential of variably treated MSC into experimental tumor environments allows efficient screening for optimizing conditions.

A complex pattern of chemokine receptor expression is seen in osteosarcoma.

BACKGROUND: Osteosarcoma is the most frequent bone tumor in childhood and adolescence. Patients with primary metastatic disease have a poor prognosis. It is therefore important to better characterize the biology of this tumor to define new prognostic markers or therapeutic targets for tailored therapy. Chemokines and their receptors have been shown to be involved in the development and progression of malignant tumors. They are thought to be active participants in the biology of osteosarcoma. The function of specific chemokines and their receptors is strongly associated with the biological context and microenvironment of their expression. In this report we characterized the expression of a series of chemokine receptors in the complex environment that defines osteosarcoma. METHODS: The overall level of chemokine receptor mRNA expression was determined using TaqMan RT-PCR of microdissected archival patient biopsy samples. Expression was then verified at the protein level by immunohistochemistry using a series of receptor specific antibody reagents to elucidate the cellular association of expression. RESULTS: Expression at the RNA level was found for most of the tested receptors. CCR1 expression was found on infiltrating mononuclear and polynuclear giant cells in the tumor. Cells associated with the lining of intratumoral vessels were shown to express CCR4. Infiltrating mononuclear cells and tumor cells both showed expression of the receptor CCR5, while CCR7 was predominantly expressed by the mononuclear infiltrate. CCR10 was only very rarely detected in few scattered infiltrating cells. CONCLUSION: Our data elucidate for the first time the cellular context of chemokine receptor expression in osteosarcoma. This is an important issue for better understanding potential chemokine/chemokine receptor function in the complex biologic processes that underlie the development and progression of osteosarcoma. Our data support the suggested involvement of chemokines and their receptors in diverse aspects of the biology of osteosarcoma, but also contradict aspects of previous reports describing the expression of these receptors in this tumor.

Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4.

The homing and tissue-specific recruitment of bone marrow-derived progenitor cells is a major issue in stem cell research and therapy. Chemokine biology plays a central role in the homing and trafficking of leukocytes. Here we show functional expression of the chemokine receptors CCR1, CCR4, CCR7, CCR10, and CXCR5 on primary isolates of CD34- mesenchymal progenitor cells as well as immortalized mesenchymal stem cell (MSC) lines. Although mRNA expression of CXCR4 was detected in both primary cells and immortalized clones, the receptor was not expressed on the cell surface. On the basis of this expression profile, the MSC could potentially home to secondary lymphatic organs (CCR7, CXCR5), skin (CCR4, CCR10), small intestine (CCR10), and salivary glands (CCR10). To study tissue-specific homing, murine CD34- MSC lines showing concordant chemokine receptor expression were either transiently labeled with CMFDA, or were stably transfected with green fluorescent protein (GFP) expression plasmids. The MSC were then injected into syngeneic healthy mice, and the distribution of the cells determined. The injected cells efficiently homed to spleen, thymus, and lymph nodes. In addition, cells were found in the mucosa of the small intestine, skin, and salivary gland. No significant recruitment to bone marrow, liver, or kidney was seen. Chemokine biology may play an important role in the homeostasis and potentially tissue recruitment of early adult progenitor cells.

In vitro and in vivo properties of a dimeric bispecific single-chain antibody IgG-fusion protein for depletion of CCR2+ target cells in mice.

The chemokine receptor CCR2 is highly expressed on leukocytes in several inflammatory diseases of both mice and men. Apart from blockade of CCR2 to prevent chemokine-dependent cell migration, depletion of CCR2(+) cells might be a promising strategy for treatment of inflammatory diseases. We therefore designed a bispecific antibody construct with the ability to deplete CCR2(+) target cells in vitro and in vivo. The bispecific antibody construct consists of two single-chain antibody variable fragments (scFv) - one recognizing murine CD3epsilon and the other recognizing murine CCR2 - joined by a short linker and fused to a modified hinge region and the C(H)2 and C(H)3 domains of murine IgG1 for dimerization. The protein was expressed in mammalian cells and purified via its C-terminal histidine tail. In vitro this construct leads to efficient antigen-specific and costimulation-independent activation of T cells and strong lysis of CCR2(+) target cells. In vivo the construct induces an almost complete depletion of CCR2(+)CD11b(+) monocytes from the peripheral blood and spleens of BALB/c mice within 24 h. This recombinant protein construct is a dimeric, bispecific antibody with markedly improved serum levels compared to conventional bispecific single-chain antibodies and the ability to deplete CCR2(+)CD11b(+) monocytes in vivo.