Masahiko Kanehira

Targeted delivery of CX3CL1 to multiple lung tumors by mesenchymal stem cells.

MSCs are nonhematopoietic stem cells capable of differentiating into various mesoderm‐type cells. MSCs have been considered to be a potential vehicle for cell‐based gene therapy because MSCs are relatively easily expanded in vitro and have the propensity to migrate to and proliferate in the tumor tissue after systemic administration. Here, we demonstrated the tropism of mouse MSCs to tumor cells in vitro and multiple tumor tissues in the lung after i.v. injection of green fluorescent protein‐positive MSCs in vivo. We transduced CX3CL1 (fractalkine), an immunostimulatory chemokine, to the mouse MSCs ex vivo using an adenoviral vector with the Arg‐Gly‐Asp‐4C peptide in the fiber knob. Intravenous injection of CX3CL1‐expressing MSCs to the mice bearing lung metastases of C26 and B16F10 cells strongly inhibited the development of lung metastases and thus prolonged the survival of these tumor‐bearing mice. This antitumor effect depended on both innate and adaptive immunity. These results suggest that MSCs can be used as a vehicle for introducing biological agents into multiple lung tumor tissues.

Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization.

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both co-culture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.

Paracrine Factors of Multipotent Stromal Cells Ameliorate Lung Injury in an Elastase-induced Emphysema Model

Multipotent stromal cells (MSCs) ameliorate several types of lung injury. The differentiation of MSCs into specific cells at the injury site has been considered as the important process in the MSC effect. However, although MSCs reduce destruction in an elastase-induced lung emphysema model, MSC differentiation is relatively rare, suggesting that MSC differentiation into specific cells does not adequately explain the recuperation observed. Humoral factors secreted by MSCs may also play an important role in ameliorating emphysema. To confirm this hypothesis, emphysema was induced in the lungs of C57BL/6 mice by intratracheal elastase injection 14 days before intratracheal MSC or phosphate-buffered saline (PBS) administration. Thereafter, lungs were collected at several time points and evaluated. Our results showed that MSCs reduced the destruction in elastase-induced emphysema. Furthermore, double immunofluorescence staining revealed infrequent MSC engraftment and differentiation into epithelial cells. Real-time PCR showed increased levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Real-time PCR and western blotting showed enhanced production of secretory leukocyte protease inhibitor (SLPI) in the lung. In-vitro coculture studies confirmed the in vivo observations. Our findings suggest that paracrine factors derived from MSCs is the main mechanism for the protection of lung tissues from elastase injury.

Mesenchymal stromal cells protect cancer cells from ROS-induced apoptosis and enhance the Warburg effect by secreting STC1.

Previous studies have demonstrated that mesenchymal stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1). This study shows that MSC-derived STC1 promotes survival of lung cancer cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more glycolytic metabolic profile. MSC-derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549 cells in an STC1-dependent manner. Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population. rSTC1-treated A549 cells displayed decreased levels of ROS, mitochondrial membrane potential (MMP), and increased lactate production, all of which were dependent on the upregulation of UCP2. Our data suggest that MSCs can promote cell survival by regulating mitochondrial respiration via STC1.

Intratracheal delivery of CX3CL1-expressing mesenchymal stem cells to multiple lung tumors.

The lung is one of the organs to which cancers from solid tumors frequently metastasize. Multiple tumors in the lung are usually treated by systemic chemotherapy because of the lack of efficient methods of targeting antitumor agents to the lung. Although intratracheal administration is an ideal route for targeting multiple lung tumors, antitumor agents are often harmful to the organ or induce inflammation. Mesenchymal stem cells (MSCs), nonhematopoietic stem cells capable of differentiating into various mesoderm-type cells, have a propensity to migrate to and proliferate in tumor tissues after systemic administration. We intratracheally injected MSCs expressing CX3CL1 (MSC/RGDFKN) into the lung of lung tumor-bearing mice with multiple metastases of C26 or Lewis lung carcinoma (LLC). Antitumor effects were evaluated by counting the number of lung metastases and survival. We demonstrated the tropism of mouse MSCs to lung tumor tissues after intratracheal administration of GFP-positive MSCs. Intratracheal injection of MSC/RGDFKN strongly inhibited growth of lung metastases of C26 or LLC, and thus prolonged survival. Intratracheal injection of MSC/RGDFKN did not induce an inflammatory reaction in the lung. These results suggest that MSCs expressing antitumor agents can be delivered intratracheally into multiple lung tumor tissues without causing inflammation.

Dendritic cells modified to express fractalkine/CX3CL1 in the treatment of preexisting tumors.

Fractalkine (CX3CL1) is a unique membrane‐bound CX3C chemokine that serves as a potent chemoattractant for lymphocytes. The hypothesis of this study is that dendritic cells (DC) genetically modified ex vivo to overexpress fractalkine would enhance the T cell‐mediated cellular immune response with a consequent induction of anti‐tumor immunity to suppress tumor growth. To prove this hypothesis, established tumors of different mouse cancer cells (B16‐F10 melanoma, H‐2b, and Colon‐26 colon adenocarcinoma, H‐2d) were treated with intratumoral injection of bone marrow‐derived DC that had been modified in vitro with an RGD fiber‐mutant adenovirus vector expressing mouse fractalkine (Ad‐FKN). In both tumor models tested, treatment of tumor‐bearing mice with Ad‐FKN‐transduced DC gave rise to a significant suppression of tumor growth along with survival advantages in the treated mice. Immunohistochemical analysis of tumors treated with direct injection of Ad‐FKN‐transduced DC demonstrated that the treatment prompted CD8+ T cells and CD4+ T cells to accumulate in the tumor milieu, leading to activation of immune‐relevant processes. Consistent with the finding, the intratumoral administration of Ad‐FKN‐transduced DC evoked tumor‐specific cytotoxic T lymphocytes, which ensued from in vivo priming of Th1 immune responses in the treated host. In addition, the anti‐tumor effect provided by intratumoral injection of Ad‐FKN‐transduced DC was completely abrogated in CD4+ T cell‐deficient mice as well as in CD8+ T cell‐deficient mice. These results support the concept that genetic modification of DC with a recombinant fractalkine adenovirus vector may be a useful strategy for cancer immunotherapy protocols.

Serial OX40 Engagement on CD4+ T Cells and Natural Killer T Cells Causes Allergic Airway Inflammation

RATIONALE OX40-OX40 ligand (OX40L) interactions have been proposed to support induction of allergic airway inflammation, which may be attributable to OX40 signaling in CD4(+) helper T cells for adaptive immune responses. However, a possible involvement of natural killer T (NKT) cells in the pathogenesis suggests that the underlying mechanisms are not yet fully elucidated. OBJECTIVES We aimed to characterize the OX40-modulated cellular contribution to allergic airway inflammation in a mouse model of house dust mite (HDM) allergen exposure. METHODS Mice were sensitized to HDM and, 3 weeks later, challenged with HDM on three consecutive days through the airways. Two days after the last exposure, bronchoalveolar lavage fluids and blood samples and lung tissues were evaluated for the airway inflammation. MEASUREMENTS AND MAIN RESULTS The development of HDM-induced eosinophilic airway inflammation was dependent on OX40 of both CD4(+) T cells and NKT cells; OX40 engagement on CD4(+) T cells in the sensitization led to pulmonary OX40L augmentation after the allergen challenge, which stimulated pulmonary NKT cells through OX40 to provide the pathogenic cytokine milieu. This was ablated by OX40L blockade by inhalation of the neutralizing antibody during the challenge, suggesting the therapeutic potential of targeting pulmonary OX40-OX40L interactions. Moreover, OX40 expression in CD4(+) T cells, but not in NKT cells, was reciprocally regulated by the helper T cell type 1-skewing transcription factor Runx3. CONCLUSIONS OX40 on not only CD4(+) T cells but also NKT cells is involved in allergic airway inflammation. Notably, pulmonary blockade of OX40 ligation on NKT cells has therapeutic implications.

Mesenchymal Stem Cells Correct Inappropriate Epithelial–mesenchyme Relation in Pulmonary Fibrosis Using Stanniocalcin-1

Current hypotheses suggest that aberrant wound healing has a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). In these hypotheses, continuous TGF-β1 secretion by alveolar epithelial cells (AECs) in abnormal wound healing has a critical role in promoting fibroblast differentiation into myofibroblasts. Mesenchymal stem cells (MSCs) home to the injury site and reduce fibrosis by secreting multifunctional antifibrotic humoral factors in IPF. In this study, we show that MSCs can correct the inadequate-communication between epithelial and mesenchymal cells through STC1 (Stanniocalcin-1) secretion in a bleomycin-induced IPF model. Inhalation of recombinant STC1 shows the same effects as the injection of MSCs. Using STC1 plasmid, it was possible to enhance the ability of MSCs to ameliorate the fibrosis. MSCs secrete large amounts of STC1 in response to TGF-β1 in comparison to AECs and fibroblasts. The antifibrotic effects of STC1 include reducing oxidative stress, endoplasmic reticulum (ER) stress, and TGF-β1 production in AECs. The STC1 effects can be controlled by blocking uncoupling protein 2 (UCP2) and the secretion is affected by the PI3/AKT/mTORC1 inhibitors. Our findings suggest that STC1 tends to correct the inappropriate epithelial-mesenchymal relationships and that STC1 plasmid transfected to MSCs or STC1 inhalation could become promising treatments for IPF.

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells.

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gain-of-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem cells.

Suppression of surfactant protein A by an epidermal growth factor receptor tyrosine kinase inhibitor exacerbates lung inflammation

Interstitial lung disease (ILD) is reported as a serious adverse event in lung cancer patients treated with gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI). However, the mechanisms of ILD associated with gefitinib remain unknown. To address the molecular mechanisms of ILD‐associated gefitinib, we determined the effect of gefitinib treatment on surfactant protein expression in vitro and in vivo. Gefitinib treatment suppressed surfactant protein (SP)‐A expression in H441 human lung adenocarcinoma cells expressing SP‐A, ‐B, ‐C and ‐D by inhibiting epidermal growth factor signal. Next, gefitinib (200 mg/kg) was given p.o. to the mice daily for 1 week. Daily administration of gefitinib gradually reduced SP‐A level in the bronchoalveolar lavage fluid. When lipopolysaccharide (LPS) was instilled intratracheally to the mice pretreated with gefitinib for 1 week, lung inflammation by LPS was exacerbated and prolonged. This exacerbation of lung inflammation was rescued by intranasal administration of SP‐A. These results demonstrated that pretreatment with gefitinib exacerbated LPS‐induced lung inflammation by reducing SP‐A expression in the lung. This study suggests that epidermal growth factor receptor tyrosine kinase inhibitor may reduce SP‐A expression in the lungs of lung cancer patients and thus patients treated with epidermal growth factor receptor tyrosine kinase inhibitor may be susceptible to pathogens. (Cancer Sci 2008; 99: 1679–1684)