Key Specifications Table
|Description||QCM ECMatrix Cell Invasion Assay, 24-well (8 µm), fluorimetric|
|Overview||Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here
Invasion through the extracellular matrix (ECM) is an important step in tumor metastasis. Cancer cells initiate invasion by adhering to and spreading along the blood vessel wall. Proteolytic enzymes, such as MMP collagenases, dissolve tiny holes in the sheath-like covering (basement membrane) surrounding the blood vessels to allow cancer cells to invade (1).
Microporous membrane inserts are widely used for cell migration and invasion assays. The most widely accepted of which is the Boyden Chamber assay. However, current methods of analysis are time-consuming and tedious, involving cotton swabbing of non-invaded cells on the topside of insert, manual staining and counting. Recently a fluorescence blocking membrane insert was introduced to address these issues; however, this approach requires labeling of the cells with Calcein-AM and extensive washing to remove free Calcein before cell invasion. The effect of this treatment on cell behavior/invasion remains questionable.
The Chemicon® QCM™ 24-well Invasion Assay does not require cell labeling, scraping, washing or counting. The 24-well insert and homogenous fluorescence detection format allows for large-scale screening and quantitative comparison of multiple samples.
In the Chemicon® QCM™ 24-well Invasion Assay, invaded cells on the bottom of the insert membrane are dissociated from the membrane when incubated with Cell Detachment Buffer. These cells are subsequently lysed and detected by the patented CyQuant GR® dye (Molecular Probes) (2-3). This green-fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids (4).
The CHEMICON® Cell Invasion Assay Kit provides an efficient system for evaluating the invasion of tumor cells through a basement membrane model. The kit utilizes ECMatrixTM, a reconstituted basement membrane matrix of proteins derived from the Engelbreth Holm-Swarm (EHS) mouse tumor (5-8). We examined the kit's performance using human fibrosarcoma (HT-1080) and non-invasive fibroblasts (NIH3T3).
The CHEMICON® Cell Invasion Assay is performed in an Invasion Chamber, based on the Boyden chamber principle. Each kit contains 24 inserts; each insert contains an 8 μm pore size polycarbonate membrane coated with a thin layer of ECMatrixTM. The ECM layer occludes the membrane pores, blocking non-invasive cells from migrating through. Invasive cells, on the other hand, migrate through the ECM layer and cling to the bottom of the polycarbonate membrane. Invaded cells on the bottom of the insert membrane are dissociated from the membrane when incubated with Cell Detachment Buffer and subsequently lysed and detected by CyQuant GR® dye.
The ability to study cell invasion through an ECM barrier, is of vital importance for developing possible metastatic inhibitors and therapeutics. The new CHEMICON® QCM™ 24-well Invasion Assay (ECM554) provides an efficient, in vitro system for quantitative analysis of tumor cell invasion.
In addition, Chemicon® continues to provide numerous migration, invasion, and adhesion products including:
· QCM™ 8μm 96-well Chemotaxis Cell Migration Assay (ECM510)
· QCM™ 5μm 96-well Chemotaxis Cell Migration Assay (ECM512)
· QCM™ 3μm 96-well Chemotaxis Cell Migration Assay (ECM515)
· QCM™ 96-well Cell Invasion Assay (ECM555)
· QCM™ 96-well Collagen-based Cell Invasion Assay (ECM556)
· 24-well Insert Cell Migration and Invasion Assay Systems
· CytoMatrix™ Cell Adhesion strips (ECM protein coated)
· QuantiMatrix™ ECM protein ELISA kits
|Materials Required but Not Delivered||1. Precision pipettes: sufficient for aliquoting cells.
2. Harvesting buffer: EDTA or trypsin cell detachment buffer. Suggested formulations include a) 2 mM EDTA/PBS, b) 0.05% trypsin in Hanks Balanced Salt Solution (HBSS) containing 25 mM HEPES, or other cell detachment formulations as optimized by individual investigators.
Note: Trypsin cell detachment buffer maybe required for difficult cell lines. Allow sufficient time for cell receptor recovery.
3. Tissue culture growth medium appropriate for subject cells, such as DMEM containing 10% FBS.
4. Chemoattractants (eg. 10% FBS) or pharmacological agents for addition to culture medium, if screening is desired.
5. Quenching Medium: serum-free medium, such as DMEM, EMEM, or FBM (fibroblast basal media), containing 5% BSA.
Note: Quenching Medium must contain divalent cations (Mg2+, Ca2+) sufficient for quenching EDTA in the harvesting buffer.
6. Sterile PBS or HBSS to wash cells.
7. Distilled water.
8. Low speed centrifuge and tubes for cell harvesting.
9. CO2 incubator appropriate for subject cells.
10. Hemocytometer or other means of counting cells.
11. Trypan blue or equivalent viability stain.
12. Fluorescence plate reader.
13. Sterile cell culture hood.
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Store kit materials at 2-8°C for up to their expiration date. Do not freeze.|
|Material Size||24 assays|
|Material Package||24 assays|
QCM ECMatrix Cell Invasion Assay, 24-well (8 µm), fluorimetric SDS
|Reference overview||Species||Pub Med ID|
|Modelling genetic and clinical heterogeneity in epithelial ovarian cancers.|
Lawrenson K, Sproul D, Grun B, Notaridou M, Benjamin E, Jacobs IJ, Dafou D, Sims AH, Gayther SA
The biology underlying early-stage epithelial ovarian cancer (EOC) development is poorly understood. Identifying biomarkers associated with early-stage disease could have a significant impact on reducing mortality. Here, we describe establishment of a three-dimensional (3D) in vitro genetic model of EOC initiation and early-stage neoplastic progression. Normal primary ovarian epithelial (POE) cells, immortalized using hTERT (immortalised ovarian epithelial [IOE] cells), were partially transformed by overexpressing the CMYC oncogene (IOE(CMYC) cells). Subsequent expression of mutant alleles of KRAS (KRAS(G12V)) or BRAF (BRAF(V600E)) created double-mutant lines (IOE(CMYC.KRAS) and IOE(CMYC.BRAF)). The transformed phenotype of IOE(CMYC) cells was further enhanced in concert with KRAS(G12V)/BRAF(V600E) expression, as in vitro analyses indicated that IOE(CMYC) cells had undergone morphological and phenotypic changes characteristic of neoplastic progression. When cultured as 3D spheroids, IOE cells underwent growth arrest, reminiscent of nonproliferative, unstimulated POE in vivo. In contrast, IOSE(CMYC+BRAF/KRAS) cells formed highly proliferative, poly-aggregate spheroid structures, showing increased expression of the Wilms tumour 1 tumourigenic marker and MIB1 proliferation marker. Transcriptomic analyses identified different gene expression profiles between the different cell lines and novel candidate genes (e.g. RGS4, CTGF and THBS1) that are somatically altered in EOCs. Gene expression signatures were compared with signatures from primary EOCs; tumours with IOE(CMYC) \'like\' signatures were more likely to be high grade (P = 0.018); tumours with BRAF signatures were associated with improved relapse-free survival (P = 0.003). In conclusion, we have established in vitro 3D models of early-stage EOCs, which reflect genetic and phenotypic heterogeneity of the disease. Molecular genetic characteristics of these models correlated with molecular and clinical features of primary EOCs.
|RNAi-mediated silencing of vEGF-C inhibits non-small cell lung cancer progression by simultaneously down-regulating the cXCR4, cCR7, vEGFR-2 and vEGFR-3-dependent axes-induced ERK, p38 and AKT signalling pathways.|
Feng Y, Hu J, Ma J, Feng K, Zhang X, Yang S, Wang W, Zhang J, Zhang Y
European journal of cancer (Oxford, England : 1990) 2011
Vascular endothelial growth factor C (VEGF-C) expression is associated with the malignant tumour phenotype making it an attractive therapeutic target. We investigated the biological roles of VEGF-C in tumour growth, migration, invasion and explored the possibility of VEGF-C as a potential therapeutic target for the treatment of non-small cell lung cancer (NSCLC). A lentivirus-mediated RNA interference (RNAi) technology was used to specifically knockdown the expression of VEGF-C in A549 cells. Quantitative reverse transcriptase-polymerase chain reaction, flow cytometry, Western blot, immunohistochemistry, cellular growth, migration, invasion and ELISA assays were used to characterise VEGF-C expression in vitro. A lung cancer xenograft model in nude mice was established to investigate whether knockdown of VEGF-C reduced tumour growth in vivo. Silencing of VEGF-C suppressed tumour cell growth, migration and invasion in vitro; suppressed tumour growth, angiogenesis and lymphangiogenesis by tail vein injection of lentivirus encoded shRNA against VEGF-C in vivo. More importantly, silencing of VEGF-C also trapped the VEGFR-2, VEGFR-3, CXCR4, CCR7-dependent axes, and down-regulated the AKT, ERK and p38 signalling pathways. These results suggest that VEGF-C has a multifaceted role in NSCLC growth, migration and invasion; that VEGF-C-mediated autocrine loops with their cognate receptors and chemokine receptors are significant factors affecting tumour progression; and that RNAi-mediated silencing of VEGF-C represents a powerful therapeutic approach for controlling NSCLC growth and metastasis.Copyright © 2011 Elsevier Ltd. All rights reserved.
|LIM and SH3 protein 1 (Lasp1) is a novel p53 transcriptional target involved in hepatocellular carcinoma.|
Bei Wang,Ping Feng,Ziwei Xiao,Ee-Chee Ren
Journal of hepatology 50 2009
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with poor prognosis associated with tumor invasion and metastasis. The tumor suppressor p53 plays critical roles in tumor development, but there is increasing evidence for its involvement in tumor metastasis with the underlying mechanisms largely unexplored.
|Screening and identification of novel B cell epitopes in human heparanase and their anti-invasion property for hepatocellular carcinoma.|
Jian-min Yang, Hui-ju Wang, Ling Du, Xiao-mei Han, Zai-yuan Ye, Yong Fang, Hou-quan Tao, Zhong-sheng Zhao, Yong-lie Zhou
Cancer immunology, immunotherapy : CII 58 1387-96 2009
BACKGROUND: The aim of this study was to screen and identify novel B cell epitopes within the human heparanase protein and to investigate the impact of self-developed anti-heparanase polypeptide antibodies on growth and invasion of HCCLM6 human hepatocellular carcinoma cells in vitro. METHODS: The flexible regions of secondary structure and the B cell epitopes of the human heparanase amino acid sequence were predicted by DNAStar and Bcepred software.The multiple antigenic peptides (MAP) of the epitopes were synthesized in eight-branched form. Rabbits were immunized with the eight-branched MAPs mixed with the universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acid 163-171). The immunogenicity of the synthesized peptides was evaluated by ELISA, western blot and immunohistochemistry. The impact of the self-developed rabbit anti-heparanase polyclonal antibodies on growth and invasion ability of HCCMLM6 cells were analyzed in a cell culture model. The cells were first treated with one of the three antibodies, respectively, and then measured by using MTT, flow cytometry, plate clone formation, invasion assay and heparan sulfate degrading enzyme assay. RESULTS: The three amino acid sequences 1-15 (MAP1), 279-293 (MAP2), and 175-189 (MAP3) in the large subunit of the human heparanase protein were predicted as its most potential epitopes. ELISA, western blot and immunohistochemistry analysis showed that all three MAPs were capable to induce high titer of serum antibodies. Antibodies induced by MAP1 and MAP2 were high specific. Furthermore, anti-MAP2 antibodies showed the strongest avidity towards liver cancer tissues. Under the treatment with the three anti-heparanase antibodies, respectively, the growth, cell cycle and clone formation of the cells remained unchanged when compared with a treatment with normal rabbit IgG. However, an inhibition of cell invasiveness and heparanase activity could be detected under the treatment with anti-MAP1- or anti-MAP2-antibody (with a terminal concentration of 100 mug/ml). The cell invasiveness was decreased by 54 and 38%, respectively, the heparanase activity by 43 and 39%, respectively. CONCLUSION: The multiple antigenic peptides MAP1 (AC 1-15) and MAP2 (AC 279-293) may be the dominant B cell epitopes in the human heparanase protein. The induced polypeptide antibodies can effectively inhibit the heparanase activity of HCCLM6 liver cancer cells and therefore influence their invasion ability, which provides a theoretic basis for the development of anti-heparanase antibodies and their clinical use as vaccine.
|Cyr61/CCN1 is a tumor suppressor in human hepatocellular carcinoma and involved in DNA damage response.|
Ping Feng,Bei Wang,Ee Chee Ren
The international journal of biochemistry & cell biology 40 2008
Cyr61/CCN1 is a secreted extracellular matrix associated protein involved in diverse biological functions and plays multiple roles in tumorigenesis. Cyr61 was down-regulated in HCC tumor tissues as observed in our previous cDNA microarray study, but its potential role in hepatocarcinogenesis is still unclear. To explore the biological significance of Cyr61 in HCC development, over-expression of this gene was established in HCC cell lines and its effects on cell proliferation, adhesion, migration and invasion were analyzed in this study. Cyr61 expression was down-regulated in HCC tumors as measured by quantitative real-time PCR and its protein level was decreased in most HCC cell lines as detected by Western blot. Over-expression of Cyr61 in HCC cell lines suppressed cell proliferation in monolayer and anchorage-independent growth in soft agar, whereas down-regulation of Cyr61 by siRNA increased cell proliferation rate. Over-expression of Cyr61 also significantly enhanced adhesion activities of HepG2 cells to various ECM proteins. Moreover, stably transfected HepG2-Cyr61 cells showed inhibited cell mobility (40-45%) and reduced invasiveness (30-40%) compared to HepG2-Neo controls. Furthermore, upon exposure to 5-Fluorouracil and UV irradiation, Cyr61 was rapidly induced in both p53(+/+) HepG2 and p53(-/-) Hep3B cells. However, only HepG2 cells showed increased G2/M phase arrest with concomitant up-regulation in p53 and p21 levels, suggesting that Cyr61 may play an active role in regulating HCC cell growth involving p53 as well as alternative pathways. In conclusion, we demonstrated that Cyr61 is a tumor suppressor in hepatocarcinogenesis and is involved in DNA damage response.
|Gonadotropin-releasing hormone agonists suppress melanoma cell motility and invasiveness through the inhibition of alpha3 integrin and MMP-2 expression and activity.|
Moretti, Roberta M, et al.
Int. J. Oncol., 33: 405-13 (2008) 2008
Cutaneous melanoma represents the leading cause of skin cancer deaths. The prognosis of highly aggressive, metastatic melanoma is still very poor, due to the resistance of the disseminated tumor to existing therapies. The clarification of the molecular mechanisms regulating melanoma growth and progression might help identify novel molecular targets for the development of new therapeutic interventions. We previously showed that gonadotropin-releasing hormone (GnRH) receptors are expressed in melanoma cells; activation of these receptors by means of GnRH agonists significantly reduces cell proliferation. In the current study, we first showed that GnRH agonists significantly reduced the metastatic behavior of melanoma cells in terms of both cell motility (haptotactic assay using laminin as the chemoattractant) and invasiveness (cell invasion assay evaluating the capacity of the cells to invade a reconstituted extracellular matrix barrier). On the basis of this observation, we then investigated the molecular mechanisms underlying the antimetastatic activity of GnRH agonists. We found that, in melanoma cells, a) the activity of the alpha3 integrin subunit is crucial for the migratory behavior of the cells; b) GnRH agonists significantly reduced alpha3 integrin expression (Western blotting and immunofluorescence studies); c) GnRH agonists significantly reduced MMP-2 expression (comparative RT-PCR) and activity (zymographic analysis performed on cell culture media). These data indicate that GnRH agonists, in addition to the previously reported antiproliferative effect, elicit a strong inhibitory activity on the migratory/invasive behavior of melanoma cells expressing GnRH receptors. These compounds reduce the metastatic potential of melanoma cells by interfering with the expression/activity of cell adhesion molecules (alpha3 integrin) and matrix metalloproteinase (MMP-2).
|Genetic regulators of large-scale transcriptional signatures in cancer|
Adler, Adam S, et al
Nat Genet, 38:421-30 (2006) 2006