carbocyanines and Neoplasm-Metastasis

Precisely locating tumors always proves to be difficult. To find a molecule that can specifically bind to tumor cells is the key. Recently, chlorotoxin (CTX) has been proved to be able to bind to many kinds of tumor cells. The CTX receptor on the cell surface has been demonstrated to be matrix metalloproteinase-2 (MMP-2). Many researchers have combined CTX with other molecules, including 131I, Cy5.5, iron oxide nanoparticles coated by polyethylene glycol (NP-PEG), and so on, and thus synthesized various types of probes that can be detected by gamma-camera, single photon emission computed tomography (SPECT) or magnetic resonance imaging (MRI). With these methods, the binding degree of CTX could be assessed. These studies demonstrated that CTX has a highly specific binding ability, high stability, and security. CTX could also inhibit or kill the tumor cells. A nonviral nanovector has been developed for gene therapy. As a result, it gradually develops into a new method of diagnosis and targeted therapy of tumors. This article reviews the current progress on CTX including the origin, chemical construction, the mechanism of binding with tumor cells, and the application to tumor imaging diagnosis and therapy.

Topics: Brain Neoplasms; Carbocyanines; Chloride Channels; Diagnostic Imaging; Ferric Compounds; Genetic Therapy; Glioma; Humans; Iodine Radioisotopes; Magnetic Resonance Imaging; Matrix Metalloproteinase 2; Nanoparticles; Neoplasm Invasiveness; Neoplasm Metastasis; Polyethylene Glycols; Scorpion Venoms; Tomography, Emission-Computed, Single-Photon

Fluorogenic probes in the near-infrared (NIR) region have the potential to provide stimuli-dependent information in living organisms. Here, we describe a new class of fluorogenic probes based on the heptamethine cyanine scaffold, the most broadly used NIR chromophore. These compounds result from modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. The resulting cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (∼170×) due to dual requirements for NIR emission: carbamate cleavage through 1,6-elimination and chromophore protonation. Illustrating their utility in complex

Topics: Animals; Carbamates; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Female; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; gamma-Glutamyltransferase; Humans; Mice; Microscopy, Confocal; Neoplasm Metastasis; Ovarian Neoplasms; Spectroscopy, Near-Infrared; Transplantation, Heterologous

The activation and execution of cancer invasion and metastasis involves a complex network of intracellular and extracellular molecule levels (such as reactive oxygen species (ROS), matrix metalloproteinases (MMPs)) and signaling cascades. Fluorescence sensing is a powerful detection tool for analytes. However, for imaging the intracellular signal cascades involving multiple molecules, traditional fluorescence probes are unsuitable, because most of them can only determine the change of species rather than response of multiple species simultaneously. Herein we constructed a novel probe: a H

Topics: Animals; Carbocyanines; Cell Line, Tumor; Color; Early Detection of Cancer; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Hydrogen Peroxide; Matrix Metalloproteinase 2; MCF-7 Cells; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Peptides; RAW 264.7 Cells

Osteosarcoma (OS) is one of most malignant bone tumors in early adolescence, which is a highly metastatic cancer and pulmonary metastasis is the most common cause of death. Thus, the development of efficient approaches to discover potential compounds that target metastasis of OS remains a topic of considerable interest. In this study, subtractive Cell-SELEX was performed to screen OS metastasis specific DNA aptamers by using cell lines with similar tumorigenic potentials but opposite metastatic aggressiveness (highly metastatic 143B cells and non-metastatic U-2 OS cells as the target and negative cells, respectively). This in vitro selection generated an ssDNA aptamer LP-16 that exhibited high binding affinity to 143B cells with an equilibrium dissociation constant (K

Topics: Animals; Aptamers, Nucleotide; Carbocyanines; Cell Line, Tumor; DNA, Single-Stranded; Female; Flow Cytometry; Fluorescent Dyes; Humans; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Probes; Neoplasm Metastasis; Osteosarcoma; SELEX Aptamer Technique

Topics: Animals; Carbocyanines; Carcinoma, Squamous Cell; Cetuximab; Disease Models, Animal; Female; Humans; Hyaluronic Acid; Mice; Neoplasm Metastasis; Optical Imaging; Ovarian Neoplasms; Pathology, Surgical; Sentinel Lymph Node; Staining and Labeling; Trastuzumab

To enhance the diagnosis of non-small cell lung cancer (NSCLC), we prepared a dual-modal probe Cy5.5-Tf-Gd-DTPA. Gd-DTPA and near-infrared (NIR) dyes were conjugated to holo-Transferrin (Tf) sequentially, the result of ICP-AES and UV showed 25 Gd ions and 1 Cy5.5 could be loaded per protein, respectively. The calculated longitudinal relaxivity R1 of Cy5.5-Tf-DTPA-Gd was 4.21 mM-1S-1 per Gd while that of Magnevist (Gd-DTPA) was only 4.02 mM-1S-1. Confocal laser scanning microscopy and immunohistochemical analyses revealed that the Cy5.5-Tf-DTPA-Gd was localized and accumulated in cytoplasmic vesicles; the cell toxicity assay showed no apparent toxicity. MR and NIR imaging of mice with subcutaneous H1299 xenografte tumors following intravenous injection of Cy5.5-Tf-DTPA-Gd revealed a strong positive contrast of the tumors, which caused a longer lasting enhancement of the MRI signal and fluorescence signal. Taken together, these studies indicate that Cy5.5-Tf-DTPA-Gd could be a good agent for MR/NIRF dual mode applications to detect both tumor in situ and its metastasis.

Topics: Animals; Carbocyanines; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Fluorescence; Fluorescent Dyes; Gadolinium; Gene Expression Regulation, Neoplastic; Humans; Ions; Lung Neoplasms; Magnetic Resonance Imaging; Magnetics; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Spectroscopy, Near-Infrared; Transferrin; Ultraviolet Rays

Tumors recruit and reprogram immune cells to support tumor development and spread, the most prominent among them being of monocytic origin such as tumor-associated macrophages (TAM) or myeloid-derived suppressor cells (MDSC). The alarmin S100A8/A9 has been implicated in the induction of TAM and MDSC. We assessed S100A9 as a molecular imaging marker for the activity of tumor-associated immune cells in a syngeneic murine breast cancer model. S100A9 could serve as a surrogate marker for tumor immune crosstalk as a function of malignancy, providing a tool with the potential for both basic research in tumor immunology and clinical stratification of patients.. BALB/c mice were inoculated with murine breast cancer cells of common origin but different metastatic capability. At different times during tumor development, optical imaging was performed using a S100A9-specific probe to visualize activated monocytes. To further explore the impact of tumor-educated monocytes, splenic myeloid cells were isolated from either healthy or tumor-bearing animals and injected into tumor-bearing mice. We analyzed the effect of the cell transfer on immune cell activity and tumor development.. We could prove S100A9-driven imaging to sensitively and specifically reflect monocyte activity in primary tumor lesions. The imaging results were corroborated by histology and fluorescence-activated cell sorting analyses. In a prospective experiment, S100A9 imaging proved indicative of the individual tumor growth, with excellent correlation. Moreover, we could show that the monocyte activity as depicted by S100A9 activity in the primary tumor lesion mirrored the tumor's metastatic behavior. Treatment with tumor-primed splenic monocytes induced increased tumor growth, accompanied by an augmented infiltration of activated myeloid cells (MDSC and TAM) into the tumor. The consecutive S100A9 expression as depicted by in vivo imaging was significantly increased.. S100A9 proved to be a sensitive and specific marker for the activity of tumor-associated immune cells. To our knowledge, S100A9 imaging represents a first in vivo imaging approach for the estimation of recruitment and activity of tumor-associated myeloid immune cells. We demonstrated the potential value of this imaging approach for prediction of local and systemic tumor development.

Topics: Animals; Calgranulin B; Carbocyanines; Cell Communication; Cell Proliferation; Diagnostic Imaging; Female; Inflammation; Leukocyte L1 Antigen Complex; Macrophages; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Monocytes; Myeloid Cells; Neoplasm Metastasis; Neoplasm Transplantation; Optics and Photonics; Rats

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood-brain and blood-tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Topics: Animals; Blood-Brain Barrier; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Deoxycytidine; Diagnostic Imaging; Drug Delivery Systems; Fluorescent Dyes; Gemcitabine; HEK293 Cells; Humans; Hypoxia; Male; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Organic Anion Transporters; Prostatic Neoplasms; Spectroscopy, Near-Infrared; Xenograft Model Antitumor Assays

Demarking lymph node networks is important for cancer staging in clinical practice. Here, we demonstrate in vivo dual-color photoacoustic lymphangiography using all-organic nanoformulated naphthalocyanines (referred to as nanonaps). Nanonap frozen micelles were self-assembled from two different naphthalocyanine dyes with near-infrared absorption at 707 nm or 860 nm. These allowed for noninvasive, nonionizing, high resolution photoacoustic identification of separate lymphatic drainage systems in vivo. With both types of nanonaps, rat lymph nodes buried deeply below an exogenously-placed 10 mm thick layer of chicken breast were clearly visualized in vivo. These results show the potential of multispectral photoacoustic imaging with nanonaps for detailed mapping of lymphatic drainage systems.

Topics: Acoustics; Animals; Breast Neoplasms; Carbocyanines; Cell Movement; Chickens; Color; Female; Humans; Lymph Nodes; Mice; Mice, Inbred BALB C; Micelles; Nanoparticles; Nanotechnology; Neoplasm Metastasis; Neoplasm Staging; Optics and Photonics; Rats; Rats, Sprague-Dawley; Sentinel Lymph Node Biopsy; Spectrophotometry

Tumor cells are inherently heterogeneous and often exhibit diminished adhesion, resulting in the shedding of tumor cells into the circulation to form circulating tumor cells (CTCs). A fraction of these are live CTCs with potential of metastatic colonization whereas others are at various stages of apoptosis making them likely to be less relevant to understanding the disease. Isolation and characterization of live CTCs may augment information yielded by standard enumeration to help physicians to more accurately establish diagnosis, choose therapy, monitor response, and provide prognosis. We previously reported on a group of near-infrared (NIR) heptamethine carbocyanine dyes that are specifically and actively transported into live cancer cells. In this study, this viable tumor cell-specific behavior was utilized to detect live CTCs in prostate cancer patients. Peripheral blood mononuclear cells (PBMCs) from 40 patients with localized prostate cancer together with 5 patients with metastatic disease were stained with IR-783, the prototype heptamethine cyanine dye. Stained cells were subjected to flow cytometric analysis to identify live (NIR(+)) CTCs from the pool of total CTCs, which were identified by EpCAM staining. In patients with localized tumor, live CTC counts corresponded with total CTC numbers. Higher live CTC counts were seen in patients with larger tumors and those with more aggressive pathologic features including positive margins and/or lymph node invasion. Even higher CTC numbers (live and total) were detected in patients with metastatic disease. Live CTC counts declined when patients were receiving effective treatments, and conversely the counts tended to rise at the time of disease progression. Our study demonstrates the feasibility of applying of this staining technique to identify live CTCs, creating an opportunity for further molecular interrogation of a more biologically relevant CTC population.

Topics: Calibration; Carbocyanines; Cell Count; Cell Line, Tumor; Cell Separation; Coloring Agents; Disease Progression; Humans; Infrared Rays; Male; Neoplasm Metastasis; Neoplastic Cells, Circulating; Prostatectomy; Prostatic Neoplasms

The ability to visualize reporter gene expression in vivo has revolutionized all facets of biologic investigation and none more so than imaging applications in oncology. Near-infrared reporter gene imaging may facilitate more accurate evaluation of chemotherapeutic response in preclinical models of orthotopic and metastatic cancers. We report the development of a cell permeable, quenched squarine probe (CytoCy5S), which is reduced by Escherichia coli nitroreductase (NTR), resulting in a near-infrared fluorescent product. Time-domain molecular imaging of NTR/CytoCy5S reporter platform permitted noninvasive monitoring of disease progression in orthotopic xenografts of disseminated leukemia, lung, and metastatic breast cancer. This methodology facilitated therapeutic evaluation of NTR gene-directed enzymatic prodrug therapy with conventional metronidazole antibiotics. These studies show NTR/CytoCy5S as a near-infrared gene reporter system with broad preclinical and prospective clinical applications within imaging, and gene therapy, of cancer.

Topics: Animals; Anti-Infective Agents; Carbocyanines; Cell Line, Tumor; Cell Survival; Escherichia coli Proteins; Green Fluorescent Proteins; HEK293 Cells; Humans; Luciferases; Magnetic Resonance Imaging; Metronidazole; Mice; Mice, Inbred NOD; Mice, SCID; Microscopy, Fluorescence; Neoplasm Metastasis; Neoplasms; Nitroimidazoles; Nitroreductases; Reproducibility of Results; Transfection; Xenograft Model Antitumor Assays

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/-) mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/-) mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/-) mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.

Topics: Animals; Antigens, CD34; Capillary Permeability; Carbocyanines; Cell Proliferation; Disease Models, Animal; Hematopoietic System; Lymphocytes, Tumor-Infiltrating; Mast Cells; Melanoma, Experimental; Mice; Models, Biological; Neoplasm Metastasis; Neoplasm Staging; Subcutaneous Tissue; Time Factors; Tumor Microenvironment

Recently, various nanoparticle systems have been developed for tumor-targeted delivery of imaging agents or drugs. However, large amount of them still have insufficient tumor accumulation and this limits their further clinical applications. Moreover, the in vivo characteristics of nanoparticles have been largely unknown, because there are few proper technologies to achieve the direct and non-invasive characterization of nanoparticles in live animals. In this paper, we determined the key factors of nanoparticles for in vivo tumor-targeting using our glycol chitosan nanoparticles (CNPs) which have proved their tumor-targeting ability in many previous papers. For this study, CNPs were labeled with near-infrared fluorescence (NIRF) dye, Cy5.5 for in vivo analysis by non-invasive optical imaging techniques. With these Cy5.5-CNPs, the factors such as in vitro/in vivo stability, deformability, and rapid uptake into target tumor cells and their effects on in vivo tumor-targeting were evaluated in various tumor-bearing mice models. In flank tumor models, Cy5.5-CNPs were selectively localized in tumor tissue than other organs, and the real-time intravascular tracking of CNPs proved the enhanced permeation and retention (EPR) effect of nanoparticles in tumor vasculature. Importantly, tumor-targeting CNPs showed an excellent tumor-specificity in brain tumors, liver tumors, and metastasis tumor models, indicating their great potential in both cancer imaging and therapy.

Topics: Animals; Blood-Brain Barrier; Carbocyanines; Cell Line; Chitosan; Materials Testing; Mice; Mice, Nude; Molecular Imaging; Molecular Structure; Nanoparticles; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Tissue Distribution

Tumor metastases occur through both the cardiovascular and lymphatic circulations. However, the majority of nanoparticle biodistribution studies have been focused on the cardiovascular circulation. In this study, we report the formulation of Cy5-labeled polylactide (Cy5-PLA) nanoparticles with controlled size and surface features and the subsequent evaluation of their lymphatic biodistribution. Cy5-PLA nanoparticles were formulated through Cy5/(BDI)ZnN(TMS)2-mediated [(BDI) = 2-((2,6-diisopropylphenyl)amido)-4-((2,6-diisopropylphenyl)-imino)-2-pentene] ring-opening polymerization of lactide followed by nanoprecipitation. Their lymphatic biodistribution was evaluated by using whole-body fluorescence imaging of nude mice and ex vivo fluorescence imaging of the resected organs. This technique has the potential for providing optical contrast and drug delivery through the lymphatic circulation for the treatment of metastatic cancer.

Topics: Animals; Carbocyanines; Drug Carriers; Drug Delivery Systems; Fluorescent Dyes; Humans; Lymphatic System; Mice; Mice, Nude; Molecular Structure; Nanoparticles; Neoplasm Metastasis; Neoplasms; Polyesters; Tissue Distribution

The breast cancer metastasis suppressor 1 (BRMS1) gene has been shown to suppress metastasis without affecting the growth of the primary tumor in mouse models. It has also been shown to suppress the metastasis of tumors derived from breast, melanoma, and, more recently, ovarian carcinoma (see ref 1). However, how BRMS1 exerts its metastasis suppressor function remains unknown. To shed light into its metastatic mechanism of action, the sensitive 2D-DIGE analysis coupled with MS has been used to identify proteins differentially expressed by either overexpressing (Mel-BRMS1) or silencing BRMS1 (sh635) in a melanoma cell line. After comparison of the protein profiles from WT, Mel-BRMS1, and sh635 cells, 79 spots were found to be differentially expressed. Mass spectrometry analysis allowed the unambiguous identification of 55 polypeptides, corresponding to 43 different proteins. Interestingly, more than 75% of the identified proteins were down-regulated in Mel-BRMS1 cells compared to WT. In contrast, all the identified proteins in sh635 cells extracts were up-regulated compared to WT. Most of the deregulated proteins are involved in cell growth/maintenance and signal transduction among other cell processes. Six differentially expressed proteins (Hsp27, Alpha1 protease inhibitor, Cofilin1, Cathepsin D, Bone morphogenetic protein receptor2, and Annexin2) were confirmed by immunoblot and functional assays. Excellent correlation was found between DIGE analysis and immunoblot results, indicating the reliability of the analysis. Available evidence on the reported functions of the identified proteins supports the emerging role of BRMS1 as negative regulator of the metastasis development. This work opens an avenue for the molecular mechanisms' characterization of metastasis suppressor genes with the aim to understand their roles.

Topics: Bone Morphogenetic Protein Receptors, Type II; Carbocyanines; Cathepsin D; Cell Line; Cell Line, Tumor; Data Interpretation, Statistical; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Humans; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Proteome; Proteomics; Repressor Proteins; RNA Interference; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin alphaVbeta3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RbetaADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for sc tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection.

Topics: Animals; Carbocyanines; Carrier Proteins; Dose-Response Relationship, Drug; Drug Administration Routes; Female; Flow Cytometry; Humans; Injections, Intra-Arterial; Integrin alphaVbeta3; Mice; Mice, Nude; Models, Biological; Neoplasm Metastasis; Ovarian Neoplasms; Peptides, Cyclic; Polymers; Radiography, Abdominal; Transfection; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

To elucidate the significance of beta-1,4-galactosyltransferase IV (beta-1,4-GT-IV) in the clinical presentation and prognostication of colorectal cancer.. Tissue lysates from paired tumor and nontumor tissues of a colon cancer patient were labeled separately with fluorescent dyes Cy5 and Cy3 for two-dimensional difference in-gel electrophoresis. Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunoblot analyses identified a down-regulated level of beta-1,4-GT-IV in the tumor tissue. In the follow-up study, paired tissue lysates were obtained from 100 colorectal cancer patients with immunoblot analyses done to compare the levels of beta-1,4-GT-IV expression in these patients.. Of 100 colorectal patients studied, 48% had down-regulated expression of beta-1,4-GT-IV in the tumor tissue but 28% of patients exhibited elevated beta-1,4-GT-IV levels. Increased beta-1,4-GT-IV in the tumor tissue was significantly coexistent with raised serum level of CA-199 and the presence of tumor metastasis (P=0.006 and P<0.001, respectively) but was independent of age and gender of patient, tumor site, tumor size, serum level of carcinoembryonic antigen, grade of tumor cell differentiation, and depth of tumor invasion. The results of logistic regression analyses suggested that tumor beta-1,4-GT-IV overexpression and tumor invasion, but not other patient variables such as tumor size and serum levels of carcinoembryonic antigen and CA19-9, were significantly correlated with the occurrence of metastases (P<0.05). In a multivariate regression analysis, the patient group with tumor beta-1,4-GT-IV overexpression strongly predicted for tumor metastasis (odds ratio, 10.009; 95% confidence interval, 2.992-33.484; P<0.001). Likewise, tumor beta-1,4-GT-IV overexpression was significantly associated with poor overall survival (P<0.01). By Cox regression analysis, this association remained significant even after adjustment for tumor metastasis (P=0.048).. Increased beta-1,4-GT-IV expression in tumor tissue was strongly associated with tumor metastases and poor prognosis in colorectal cancer.

Topics: Aged; Carbocyanines; Colorectal Neoplasms; Electrophoresis, Gel, Two-Dimensional; Female; Galactosyltransferases; Humans; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Risk Factors; Up-Regulation