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Background The existing shortage of animal models that properly mimic the progression of early-stage human lung cancer from a solitary confined tumor to an invasive metastatic disease hinders accurate characterization of key interactions between lung cancer cells and their stroma. We herein describe a novel orthotopic animal model that addresses these concerns and consequently serves as an attractive platform to study tumor–stromal cell interactions under conditions that reflect early-stage lung cancer. Methods Unlike previous methodologies, we directly injected small numbers of human or murine lung cancer cells into murine's left lung and longitudinally monitored disease progression. Next, we used green fluorescent protein-tagged tumor cells and immuno-fluorescent staining to determine the tumor's microanatomic distribution and to look for tumor-infiltrating immune cells and stromal cells. Finally, we compared chemokine gene expression patterns in the tumor and lung microenvironment. Results We successfully generated a solitary pulmonary nodule surrounded by normal lung parenchyma that grew locally and spread distally over time. Notably, we found that both fibroblasts and leukocytes are recruited to the tumor's margins and that distinct myeloid cell attracting and CCR2-binding chemokines are specifically induced in the tumor microenvironment. Conclusion Our orthotopic lung cancer model closely mimics the pathologic sequence of events that characterizes early-stage human lung cancer propagation. It further introduces new means to monitor tumor–stromal cell interactions and offers unique opportunities to test therapeutic targets under conditions that reflect early-stage lung cancer. We argue that for such purposes our model is superior to lung cancer models that are based either on genetic induction of epithelial transformation or on ectopic transplantation of malignant cells.

Background Patients with kidney failure frequently require the formation of an arterio-venous fistula (AVF) in which a vein is connected to an artery resulting in arterialization of the vein to allow adequate blood flow into an external ‘artificial kidney’. In most patients, neo-intimal hyperplasia (NIH) ensues, causing narrowing and subsequent occlusion of the vein, leading to significant morbidity. The cellular events causing venous NIH may serve as ideal targets for molecular-based therapies. However, therapeutic gene delivery into the vascular system is seriously impeded by problems related to the low efficacy and toxicity of targeted viral vector delivery. Materials and methods To explore the feasibility of a plasmid-based vascular gene delivery system, we established a rat AVF model that develops NIH. Plasmids encoding for reporter or therapeutic genes were delivered into the blood vessels at the time or after AVF formation. Results Intra-luminal injection of plasmid into the AVF resulted in extensive and long-term reporter gene expression at the venous limb mainly at the site of NIH formation. Transgene expression was confined to endothelial cells and myofibroblasts that migrate inwards from the adventitia and form the NIH lesion. There was no detrimental tissue reaction to plasmid delivery, contrasting with the severe inflammatory response observed after adenovirus infection. Intra-vascular delivery of a plasmid carrying the endothelial nitric oxide synthase gene resulted in sustained production of nitric oxide, previously shown to mitigate NIH formation. Conclusions These findings open the possibility of vascular transduction with naked DNA bearing therapeutic genes in areas prone to NIH to ameliorate vein graft pathologies using simple and clinically applicable vector delivery methods. Copyright © 2011 John Wiley & Sons, Ltd.

Objectives Carcinoma-associated fibroblasts are reported to communicate microenvironment-derived signals through chemokine/chemokine receptor interaction, influencing carcinogenesis. We sought to characterize roles of CXCL12/CXCR4 in crosstalk between non–small cell lung cancer epithelial cell and carcinoma-associated fibroblasts and in tumor growth. Methods Non–small cell lung cancer tumor samples obtained at surgery and from tumor arrays, as well as primary carcinoma-associated fibroblast and epithelial cell lines generated from fresh tumors, were assessed for CXCL12/CXCR4 expression, tissue localization, and production. Colony assays, extracellular signal–regulated kinase signaling, and chemokine production were measured to assess cancer cell responsiveness to CXCL12 stimulation with or without CXCR4 antagonists. Results CXCL12 and CXCR4 were detected in all major subtypes of non–small cell lung cancer. CXCL12-expressing carcinoma-associated fibroblasts were mostly located near CXCL12-negative tumor cells, whereas CXCL12-positive tumor cells were mostly surrounded by CXCL12-negative stroma. Intratumoral CXCL12 levels were significantly higher than serum levels. CXCL12 expression correlated with advanced disease stage. In vitro, tumor cell lines produced variable amounts of CXCL12 and expressed high levels of CXCR4. Carcinoma-associated fibroblasts cell lines produced high amounts of CXCL12 and expressed variable levels of CXCR4. Stimulation of non–small cell lung cancer neoplastic cells with CXCL12 increased colony-forming capacity, induced extracellular signal–regulated kinase phosphorylation, and production of the proinflammatory chemokine CCL20. CXCR4 antagonists attenuated these effects. Conclusions Interaction between carcinoma-associated fibroblasts and tumor epithelial cells through the CXCL12/CXCR4 axis plays a role in non–small cell lung cancer tumor proliferation, marking this axis as a target for immune intervention.

GNE Myopathy (GNEM) is a neuromuscular disorder caused by mutations in the GNE gene. It is a slowly progressive distal and proximal muscle weakness sparing the quadriceps. In this study, we applied our model of mutated M743T GNE enzyme skeletal muscle-cultured myoblasts and paired healthy controls to depict the pattern of signaling proteins controlling survival and/or apoptosis of the PI3K/AKT, BCL2, ARTS/XIAP pathways, examined the effects of metabolic changes/stimuli on their expression and activation, and their potential role in GNEM. Immunoblot analysis of the GNEM myoblasts indicated a notable increased level of activated PTEN and PDK1 and a trend of relative differences in the expression and activation of the examined signaling molecules with variability among the cultures. ANOVA analysis showed a highly significant interaction between the level of PTEN and the patients groups. In parallel, the interaction between the level of BCL2, BAX and PTEN with the specific PI3K/AKT inhibitor-LY294002 was highly significant for BCL2 and nearly significant for PTEN and BAX. The pattern of the ARTS/XIAP signaling proteins of GNEM and the paired controls was variable, with no significant differences between the two cell types. The response of the GNEM cells to the metabolic changes/stimuli: serum depletion and insulin challenge, as indicated by expression of selected signaling proteins, was variable and similar to the control cells. Taken together, our observations provide a clearer insight into specific signaling molecules influencing growth and survival of GNEM muscle cells.

Patients with kidney failure frequently require the formation of an arterio-venous fistula (AVF) in which a vein is connected to an artery resulting in arterialization of the vein to allow adequate blood flow into an external 'artificial kidney'. In most patients, neo-intimal hyperplasia (NIH) ensues, causing narrowing and subsequent occlusion of the vein, leading to significant morbidity. The cellular events causing venous NIH may serve as ideal targets for molecular-based therapies. However, therapeutic gene delivery into the vascular system is seriously impeded by problems related to the low efficacy and toxicity of targeted viral vector delivery.To explore the feasibility of a plasmid-based vascular gene delivery system, we established a rat AVF model that develops NIH. Plasmids encoding for reporter or therapeutic genes were delivered into the blood vessels at the time or after AVF formation.Intra-luminal injection of plasmid into the AVF resulted in extensive and long-term reporter gene expression at the venous limb mainly at the site of NIH formation. Transgene expression was confined to endothelial cells and myofibroblasts that migrate inwards from the adventitia and form the NIH lesion. There was no detrimental tissue reaction to plasmid delivery, contrasting with the severe inflammatory response observed after adenovirus infection. Intra-vascular delivery of a plasmid carrying the endothelial nitric oxide synthase gene resulted in sustained production of nitric oxide, previously shown to mitigate NIH formation.These findings open the possibility of vascular transduction with naked DNA bearing therapeutic genes in areas prone to NIH to ameliorate vein graft pathologies using simple and clinically applicable vector delivery methods.

OBJECTIVES: Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth. METHODS: A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation. RESULTS: CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52-15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells. CONCLUSION: Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.

Hereditary inclusion body myopathy (HIBM) is a unique muscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. GNE encodes a bi-functional enzyme acting in the biosynthetic pathway of sialic acid. Since the underlying myopathological mechanism leading to the disease phenotype is poorly understood, we have established human myoblasts cultures, derived from HIBM satellite cells carrying the homozygous M712T mutation, and identified cellular and molecular characteristics of these cells. HIBM and control myoblasts showed similar heterogeneous patterns of proliferation and differentiation. Upon apoptosis induction, phosphatidylserine externalization was similar in HIBM and controls. In contrast, the active forms of caspase-3 and -9 were strongly enhanced in most HIBM cultures compared to controls, while pAkt, downregulated in controls, remained high in HIBM cells. These results could indicate impaired apoptotic signaling in HIBM cells. Since satellite cells enable partial regeneration of the post-mitotic muscle tissue, these altered processes could contribute to the muscle mass loss seen in patients. The identification of survival defects in HIBM affected muscle cells could disclose new functions for GNE in muscle cells.

Objectives CXCR4/CXCL12 interactions promote non–small cell lung cancer (NSCLC) growth and dissemination. Furthermore, this axis might promote NSCLC resistance to chemotherapy and/or radiotherapy. Therefore, the CXCR4/CXCL12 axis constitutes an attractive therapeutic target for the treatment of NSCLC. We aimed to characterize the therapeutic efficacy of the novel CXCR4 antagonist BKT140 against human NSCLC. Methods We determined the CXCR4 expression in 5 NSCLC cell lines (H358, A549, H460, H1299, and L4). We then tested the colony-forming capacity and proliferation of these cells in the presence of CXCL12 and BKT140. Next, we measured the in vivo growth of A549 and H460 xenografts with or without BKT140 treatment. Finally, we examined, in vitro, the potential antiproliferative effect of BKT140 combined with cisplatin or paclitaxel and after irradiation of NSCLC cells. Results All tested cell lines expressed CXCR4 and showed increased colony formation in response to CXCL12 stimulation. BKT140 reduced the colony-forming capacity of NSCLC cells. Proliferation assays demonstrated both cytotoxic and cytostatic properties for this peptide. H460 cells were the most sensitive to BKT140 and A549 cells the least. Subcutaneous administration of BKT140 significantly delayed the development of H460 xenografts and showed a similar trend for A549 xenografts. Finally, the antiproliferative effects of BKT140 appears to be additive to those of chemotherapeutic drugs and radiotherapy. Conclusions Targeting the CXCL12/CXCR4 axis with BKT140 attenuated NSCLC cells tumor growth and augmented the effects of chemotherapy and radiotherapy. Future research will benefit from delineating the downstream mechanism of BKT140 action and defining BKT140 susceptibility markers.