C. Blechet

Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion.

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells.

Monitoring of tumour progression using bioluminescence imaging and computed tomography scanning in a nude mouse orthotopic model of human small cell lung cancer

Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.

Kallikrein-Related Peptidase 6 Overexpression Promotes Non-Small Cell Lung Cancer Cell Proliferation and is associated with poor patient outcome

Introduction The human kallikrein-related peptidases (KLK) are a family of serine proteases that are often aberrantly expressed in common human malignancies and contribute to neoplastic progression through multifaceted roles. Methods We evaluated KLK6 expression in the tumoral and normal adjacent lung tissue of 56 patients with Non-Small Cell Lung Cancer (NSCLC) by real-time RT-PCR and immunohistochemistry. To determine the impact of KLK6 overexpression on the growth of lung cancer cells, we integrated the cDNA encoding the complete sequence of KLK6, through homolog recombination, in a NSCLC line (A549 Flp-In) and determined the growth rate of two independent clones. Progression of the KLK6- and parental cells inside the cell cycle was assessed by flow cytometry following synchronization of cells at the end of the G1 phase with starvation and hydroxyurea treatments. Key regulator proteins of the cell cycle were analyzed by Western blot in synchronized and unsynchronized cells. Results We found KLK6 transcript up-regulation in tumor tissues from patients with NSCLC and association of KLK6 status with low patient survival. KLK6 immunoreactivity was restricted to epithelial cells of normal bronchi and detected in most of cancer samples, in which KLK6 signal intensity correlated with well differentiated tumors. Ectopic expression of KLK6 dramatically enhanced NSCLC cell growth. Analysis of cell cycle progression revealed that promotion of cell growth caused by KLK6 results from an acceleration of cell cycle progression through G1/S transition, which was accompanied with a marked increase of cyclin E and repression of p21. In addition, expression of KLK6 in NSCLC cells was associated with an increase of c-Myc that is well-know to promote cell-cycle progression via regulation of cyclin D/E activation and down-regulation of p21. Conclusion ectopic expression of KLK6 facilitates cell cycle progression, certainly through alteration of c-Myc and downstream key regulators, and thus promotes cell proliferation. Moreover, KLK6 is overexpressed in NSCLC and associated with poor prognosis. Altogether, those findings suggest that KLK6 might play a central role in NSCLC development and progression.

Monitoring of tumor progression using bioluminescence imaging in a nude mice orthotopic model of human small cell lung cancer

Introduction Lung cancer is the main cause of cancer death throughout the world and a clinically relevant animal model of human small cell lung carcinoma (SCLC) should be useful to study the molecular aspects of the tumor progression and test the efficiency of new therapeutic agents. In this study, we generated a reproductible and reliable nude mice orthotopic model of human SCLC based on NCI-H209 tumor cells genetically modified to express firefly luciferase. Methods NCI-H209 cells were transfected with pCMV-luc plasmid and clones highly expressing luciferase were isolated and amplified. Cells were analyzed for long-term bioluminescent stability and a clone was subcutaneously passaged twice in vivo to enhance tumorigenicity. Cells resuspended in Matrigel ® and/or EDTA RPMI medium containing a Tc 99M colloid were implanted intrabronchially using a catheter inserted into the trachea and positioned into the right main bronchus using interventional imaging. Punctual deposition of cells was then assessed by scintigraphy. Tumor progression was then followed using bioluminescence imaging. Results Only tumor nodules were observed into lung and trachea when cells were implanted with EDTA Lung tumor invading parenchyma were present in 40% of the mice with Matrigel ® and improved to 75% with EDTA and Matrigel ® . The growth of the primary tumor was sensitively and non-invasively followed and quantified by bioluminescence imaging using a CCD-camera. This tool allows a real-time monitoring of tumor progression on the same animals over a 2-12 week period. Combination of 3D bioluminescence imaging and computed tomography scanning was used to further document tumor location and measurement. Subsequently, the histological analysis of tissue sections confirmed the presence of a lung tumor. Conclusion Our nude mice orthotopic model resembles various stages human small cell lung carcinoma, and then could be useful for evaluating new therapeutic strategies.