Lung Cancer Metastasis Model Research Articles

An Informatics-assisted Label-free Quantitation Strategy that Depicts Phosphoproteomic Profiles in Lung Cancer Cell Invasion

Aberrant protein phosphorylation plays important roles in cancer-related cell signaling. With the goal of achieving multiplexed, comprehensive, and fully automated relative quantitation of site-specific phosphorylation, we present a simple label-free strategy combining an automated pH/acid-controlled IMAC procedure and informatics-assisted SEMI (sequence, elution time, mass-to-charge, and internal standard) algorithm. The SEMI strategy effectively increased the number of quantifiable peptides more than 4-fold in replicate experiments (from 262 to 1171, p < 0.05, false discovery rate = 0.46%) by using a fragmental regression algorithm for elution time alignment followed by peptide cross-assignment in all LC-MS/MS runs. In addition, the strategy demonstrated good quantitation accuracy (10-12%) for standard phosphoprotein and variation less than 1.9 fold (within 99% confidence range) in proteome scale and reliable linear quantitation correlation (R(2) = 0.99) with 4000-fold dynamic concentrations, which was attributed to our reproducible experimental procedure and informatics-assisted peptide alignment tool to minimize system variations. In an attempt to explore metastasis-associated phosphoproteomic alterations in lung cancer, this approach was used to delineate differential phosphoproteomic profiles of a lung cancer metastasis model. Without sample fractionation, the SEMI algorithm enabled quantification of 1796 unique phosphopeptides (false discovery rate = 0.56%) corresponding to 854 phosphoproteins from a series of non-small cell lung cancer lines with varying degrees of in vivo invasiveness. Nearly 40% of the phosphopeptides showed >2-fold change in highly invasive cells; validation of phosphoprotein subsets by Western blotting not only demonstrated the consistency of data obtained by our SEMI strategy but also revealed that such dramatic changes in the phosphoproteome result mostly from translational or post-translational regulation. Mapping of these differentially expressed phosphoproteins in multiple cellular pathways related to cancer invasion and metastasis suggests that the site and degree of phosphorylation might have distinct patterns or functions in the complex process of cancer progression.

Administration of PLK-1 small interfering RNA with atelocollagen prevents the growth of liver metastases of lung cancer

Liver metastasis is one of the most important prognostic factors in lung cancer patients. However, current therapies are not sufficient. RNA interference provides us a powerful and promising approach for treating human diseases including cancers. Herein, we investigated the in vitro effects of PLK-1 small interfering RNA (siRNA) on human lung cancer cell lines and the in vivo usage of PLK-1 siRNA with atelocollagen as a drug delivery system in a murine liver metastasis model of lung cancer. PLK-1 was overexpressed in cell lines and in cancerous tissues from lung cancer patients. PLK-1 siRNA treatment inhibited growth and induced apoptosis in a concentration-dependent manner. To verify in vivo efficacy, we confirmed that atelocollagen was a useful drug delivery system in our model of implanted luciferase-labeled A549LUC cells by detecting reduced bioluminescence after an i.v. injection of luciferase GL3 siRNA/atelocollagen. PLK-1 siRNA/atelocollagen was also successfully transfected into cells and inhibited the progression of metastases. This study shows the efficacy of i.v. administration of PLK-1 siRNA/atelocollagen for liver metastases of lung cancer. We believe siRNA therapy will be a powerful and promising strategy against advanced lung cancer.

Effects of Feiji Formula on lung cancer metastasis in mice

To observe the effects of Feiji Formula, a compound traditional Chinese herbal medicine, on lung cancer metastasis in mice. The lung cancer metastasis model of mice was established in this experiment study. Twenty-four mice were randomly divided into three groups: untreated group, cisplatin group and Feiji Formula group. Mice in the Feiji Formula group were treated with Feiji Formula decoction; in cisplatin group, with cisplatin by intraperitoneal injection; and in the untreated group, with normal saline (NS). After twenty-day treatment, the body and tumor weights as well as the number of metastatic tumors in both lungs of each mouse were measured. The body weight of mice in cisplatin group was significantly less than that of Feiji Formula group and untreated group (P<0.01); the tumor weight of mice in cisplatin group and Feiji Formula group was markedly lower than that of untreated group (P<0.01); and the number of metastatic tumors in cisplatin group and Feiji Formula group was markedly lower than that of the untreated group (P<0.01), no significant difference between the Feiji Formula group and cisplatin group in terms of the weights and the numbers of metastatic tumors in bilateral lungs. Feiji Formula can suppress tumor growth and decrease the number of lung metastatic tumors in the mice, and maintain the body weight of the mice.

A double three‐step theory of brain metastasis in mice: the role of the pia mater and matrix metalloproteinases

The brain is frequently affected by the spread of lung cancer, and haematogenous metastasis is a common route to brain metastasis. We therefore developed an isogenic brain metastasis model of lung cancer to use the Lewis lung carcinoma cell line and analysed dynamics of neoplastic cells after extravasation. Histological analysis revealed two characteristic patterns: metastatic foci exhibiting an angiocentric pattern were designated 'perivascular proliferations'; neoplastic cells infiltrating the brain parenchyma were designated 'invasive proliferations'. Electron microscopic observation of perivascular proliferations showed that neoplastic cells were confined to the perivascular space. In invasive proliferations, however, fragments of collagen fibre were observed in the gaps between neoplastic cells, indicating that the neoplastic cells had disintegrated the pia-glial membrane. We analysed the expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 by using both immunohistochemical analysis and real-time polymerase chain reaction analysis. MMP-2 expression was significantly higher in invasive proliferations. MMP-9 expression was significantly higher in day 7, but there was no significant difference in day 11. The pia-glial membrane and perivascular space are the barriers that neoplastic cells must overcome to infiltrate the brain. In conclusion, our findings suggest that brain metastasis requires two distinct processes.

Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC‐5) cells

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.

780. A Novel Multi-Component Molecular Conjugate Vector for Highly Efficient p53-Mediated Tumor-Specific Gene Therapy

Top of pageAbstract In an attempt to overcome the individual hurdles inherent in non viral gene delivery, we have generated a targeting multi-component gene therapy vector that has advantages over each modality administered as a mono-therapy. Taking advantage of the Simian Virus (SV) 40 Nuclear Localization Signaling (NLS) peptide as well as its DNA Nuclear Targeting Signal (DNTS), we have developed a polyethylenimine (PEI)-based multi-component therapeutic gene delivery vector to serve as a proof of principle in increasing tumor-specific gene delivery. In this novel vector, the high affinity and specificity of Peptide Nucleic Acid (PNA) binding to its complementary sequence was also utilized to bind NLS peptide to plasmid DNA containing the DNTS, PNA binding site and either bgal or the p53 gene. Furthermore, the inclusion of PEG in the vector reduced the steric hindrance between the components and ensured a more stable and efficient delivery of the complex to the tumor cells. This newly developed vector was up to a 40-fold more efficient than our previously reported CNGRC-mediated vector which targets aminopeptidase N/CD13 in a nude mouse model (Human Gene Therapy, 2005, in press) and J591 mAb-mediated vector which targets PSMA in an orthotopic mouse model (Molecular Therapy, 2005, in press), when administered systemically to mice bearing H1299 (non-small cell lung cancer) subcutaneous tumors. The targeting polyplex, CNGRC/PEG/PEI/DNA-DNTS-bgal/NLS, expressing bgal, also showed as much as a 100-fold increase in transduction over our previously reported CD13-targeted vector in vitro and up to 10-fold increase in gene delivery over either CNGRC/PEG/PEI/DNA-DNTS-bgal or CNGRC/PEG/PEI/DNA-bgal/NLS alone, indicating the additive effects of the NLS motifs in gene delivery. Examination of nuclear localization as an indicator of gene expression in H1299 cells showed CNGRC/PEG/PEI/DNA-DNTS-bgal/NLS conjugates in the cell nucleus but not the conjugates without NLS signals. Furthermore, a 40-fold increase in in-vivo gene delivery to tumors over our previously reported CNGRC-targeted vector resulted in a significant (80%) reduction in tumor volumes over a 21-day period when bgal was replaced with p53 as the therapeutic gene in the targeting vector, thereby showing a very promising efficacy for systemic p53 gene therapy in a human lung cancer metastasis model. An increase in gene delivery with the therapeutic targeting vector, CNGRC/PEG/PEI/DNA-DNTS-p53/NLS, was corroborated by a more robust p53 mRNA and gene expression in tumors and stained tumor sections as compared to other organs from the same mice or mice injected with vector without the NLS motifs. Pharmacologic and toxicity studies showed no marked adverse effects on major organs. This |[ldquo]|artificial virus|[rdquo]| we have developed here certainly needs further optimization as a potentially suitable multi-component delivery system in overcoming most of the current hurdles in non viral gene delivery. We are currently in the process of extensive evaluation of this novel vector for use in p53 clinical trials.

Bone metastasis model with multiorgan dissemination of human small-cell lung cancer (SBC-5) cells in natural killer cell-depleted SCID mice.

Lung cancer is commonly associated with multiorgan metastasis, and bone is a frequent metastatic site for lung cancer. Nevertheless, no bone metastasis model of lung cancer with multiorgan dissemination is available, which could provide opportunity to study the molecular pathogenesis. We examined the abilities of eight human lung cancer cell lines injected intravenously into natural killer (NK) cell-depleted SCID mice to generate metastatic nodules in bone and multiple organs, and explored the correlation of the parathyroid hormone-related protein (PTHrP) with the bone metastasis. Although all the small-cell carcinoma cell lines (SBC-5, SBC-3, SBC-3/ADM, H69, H69/VP) formed metastatic nodules in multiple organs (liver, kidney, and lymph nodes), only SBC-5 cells reproducibly developed bone metastases. Squamous cell carcinoma (RERF-LC-AI) cells metastasized mainly into the liver and kidneys, whereas adenocarcinoma (PC-14, A549) mainly produced colonies in the lungs. As assessed by X-ray photography, the osteolytic bone metastases produced by SBC-5 cells were detected as early as on day 28, and all recipient mice developed bone metastasis by day 35. The expression of PTHrP in eight cell lines was directly correlated with the formation of bone metastasis. No correlation was observed between the formation of bone metastasis and the expression of other metastasis-related cytokines (IL-1, IL-6, IL-8, IL-10, IL-11, TNF-alpha, VEGF, M-CSF). Consistent with the formation of bone metastasis by SBC-5 cells, the levels of PTHrP and calcium in the mouse serum were increased in a time-dependent manner, suggesting that PTHrP produced by human lung cancer may play a crucial role in the formation of bone metastasis and hypercalcemia. These findings indicate that a bone metastasis model of SBC-5 cells may be useful for clarifying the molecular aspects of the metastatic processes in different organ microenvironments and the development of therapeutic modalities for lung cancer patients with bone metastases.