Key Specifications Table
|Description||QCM™ Collagen Cell Invasion Assay, 24-well (8 µm), Colorimetric|
|Overview||NEW!! High Sensitivity Collagen Invasion Assay! 24-well (8 µm), Colorimetric Click Here
Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here
Penetration of the subendothelial basement membrane marks a critical turning point in the metastatic process. As proliferating neoplastic cells attempt to escape the primary tumor site, local invasion of the surrounding tissue (interstitial stroma) must occur. Neovascularization is initiated by expression of angiogenic factors (e.g. FGF, VEGF, HGF), providing nutritional requirements and access to the vascular system. Prior to penetrating the blood vessel endothelium and gaining access to the blood stream (intravasation), cancer cells must invade local tissues by degrading ECM components and ultimately, transverse the basement membrane. Once in circulation, these cells can form metastatic colonies at secondary locations, making this membrane a key invasive barrier.
The basement membrane surrounding the blood vessel endothelium is a thin, specialized network of extracellular matrix proteins (ECM) that serves many functions. Comprised of proteins and proteoglycans, such as collagen, laminin, entactin, fibronectin, heparin sulfate and perlecan, this membrane acts as a physical barrier between the epithelium and underlying tissues. It provides cell surface anchorage (via integrins, receptor kinases, and cell surface proteoglycans), induces cellular differentiation, gives architectural support, and limits the migration of normal cells. The ability of tumor cells to degrade the ECM components of the basement membrane and surrounding tissues is directly correlated with metastatic potential. By releasing proteolytic enzymes (e.g. MMP collagenases, plasminogen activators, cathepsins), cancer cells are able to breach the membrane and penetrate the blood vessel wall. Collagen, the primary structural element of the basement membrane and tissue scaffolding protein, represents the main deterrent in the migration of tumor cells.
The ability to study cell invasion through a collagen barrier, is of vital importance for developing possible metastatic inhibitors and therapeutics. The new CHEMICON QCM™ 24-well Collagen-based Invasion Assay (ECM551) provides an efficient, in vitro system for quantitative analysis of tumor cell invasion.
The CHEMICON QCM™ 24-well Collagen-based Invasion Assay (ECM551) eliminates cell pre-labeling and manual counting. The 24-well insert and colorimetric detection format allows for quantitative comparison of multiple samples. ECM551 uses purified chicken type I collagen as a matrix. The product is 99.9% pure native atelomeric avian collagen. Approximately 85% type I and 15% type III collagen protein. The material has been specially prepared to maintain its native state as much as possible. The telomeric ends of the collagen I have been enzymatically removed. Since most of the species identity is found within the telomeric ends, our atelomeric chicken collagen should provide a suitable substrate for most any animal species. The working concentration and precise purification protocol are proprietary and will not be disclosed, however it at least a 0.3% solution of treated avian collagen.
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
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 polymerized collagen. The collagen layer occludes the membrane pores, blocking non-invasive cells from migrating through. Invasive cells, on the other hand, migrate through the polymerized collagen layer and cling to the bottom of the polycarbonate membrane. Invaded cells on the bottom of the insert membrane are incubated with Cell Stain Solution, then subsequently extracted and detected on a standard microplate reader (560 nm).
|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. Microplate reader (560 nm).
13. 24-well tissue culture plate.
14. 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 wells|
|Material Package||24 wells|
|Reference overview||Pub Med ID|
|MiR-145 inhibits tumor angiogenesis and growth by N-RAS and VEGF. |
Chao Zou,Qing Xu,Feng Mao,Dan Li,Chuanxiu Bian,Ling-Zhi Liu,Yue Jiang,Xiaona Chen,Yanting Qi,Xiaolong Zhang,Xuejing Wang,Qiang Sun,Hsiang-Fu Kung,Marie C Lin,Andreas Dress,Fiona Wardle,Bing-Hua Jiang,Lihui Lai
Cell cycle (Georgetown, Tex.) 11 2012
MiR-145 is known as a tumor suppressor in numerous human cancers. However, its role in tumor angiogenesis remains poorly defined. In this study, we found that miR-145 was significantly downregulated in breast cancer tissues by using 106 cases of normal and cancer tissues as well as in breast cancer cells. MiR-145 exhibited inhibitory role in tumor angiogenesis, cell growth and invasion and tumor growth through the post-transcriptional regulation of the novel targets N-RAS and VEGF-A. In addition, we provide evidence that the expression levels of miR-145 correlate inversely with malignancy stages of breast tumors, although there is no association between miR-145 levels and hormone receptor levels in breast cancer. Taken together, these results demonstrate that miR-145 plays important inhibitory role in breast cancer malignancy by targeting N-RAS and VEGF-A, which may be potential therapeutic and diagnostic targets.
|Lyn is involved in CD24-induced ERK1/2 activation in colorectal cancer. |
Ning Su,Liang Peng,Bingqing Xia,Yingying Zhao,Angao Xu,Jing Wang,Xinying Wang,Bo Jiang
Molecular cancer 11 2012
|Plumbagin, a plant derived natural agent inhibits the growth of pancreatic cancer cells in in vitro and in vivo via targeting EGFR, Stat3 and NF-κB signaling pathways. |
Bilal Bin Hafeez,Mohammad Sarwar Jamal,Joseph W Fischer,Ala Mustafa,Ajit Kumar Verma
International journal of cancer. Journal international du cancer 131 2012
Pancreatic cancer (PC) is the most aggressive malignant disease, ranks as the fourth most leading cause of cancer-related death among men and women in the United States. We present here that plumbagin (PL), a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L, inhibits the growth of PC cells both in vitro and in vivo model systems. PL treatment induces apoptosis and inhibits cell viability of PC cells (PANC1, BxPC3 and ASPC1). In addition, i.p. administration of PL (2 mg/kg body weight, 5 days a week) in severe combined immunodeficiency (SCID) mice beginning 3 days after ectopic implantation of PANC1 cells resulted in a significant (P < 0.01) inhibition of both tumor weight and volume. PL treatment inhibited (1) constitutive expression of epidermal growth factor receptor (EGFR), pStat3Tyr705 and pStat3Ser727, (2) DNA binding of Stat3 and (3) physical interaction of EGFR with Stat3, in both cultured PANC1 cells and their xenograft tumors. PL treatment also inhibited phosphorylation and DNA-binding activity of NF-κB in both cultured PC cells (PANC1 and ASPC1) and in PANC1 cells xenograft tumors. Downstream target genes (cyclin D1, MMP9 and Survivin) of Stat3 and NF-κB were similarly inhibited. These results suggest that PL may be used as a novel therapeutic agent against human PC. Published 2012 Wiley-Liss, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America.
|TRAIL-induced caspase/p38 activation is responsible for the increased catalytic and invasive activities of Akt. |
Bo K Sun,Joo-Hang Kim,Hoan N Nguyen,So Y Kim,Seeun Oh,Yong J Lee,Jae J Song
International journal of oncology 38 2011
We previously observed that TRAIL induces acquired TRAIL resistance coinciding with increased Akt phosphorylation brought about by the Src-PI3K-Akt signaling pathways and mediated by c-Cbl. c-Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is simultaneously involved in the rapid degradation of TRAIL receptors and Akt phosphorylation during TRAIL treatment. Here, we show that Akt phosphorylation is not exclusively responsible for acquired TRAIL resistance. Akt catalytic activation is known to increase during metabolic oxidative stress, but we show that TRAIL also dramatically induces the catalytic activation of Akt in TRAIL-sensitive cells, but not in TRAIL-resistant cells. This suggests that Akt catalytic activation during TRAIL-induced apoptosis is likely to play a compensatory role in the maintenance of cell homeostasis. In addition, activated p38 and phosphorylated HSP27 were found to act as downstream effector molecules of p38 during TRAIL treatment and were shown to be responsible for increased Akt catalytic and invasive activities.Full Text Article
|Inhibition of adhesion, invasion, and metastasis by antibodies targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen). |
Rosalyn D Blumenthal,Hans J Hansen,David M Goldenberg
Cancer research 65 2005
CEACAM5 and CEACAM6 are overexpressed in many cancers and are associated with adhesion and invasion. The effects of three monoclonal antibodies targeting different epitopes on these antigens (NH2-terminal [MN-3] and A1B1 domains [MN-15] shared by CEACAM5 and CEACAM6 and the A3B3 domain [MN-14] restricted to CEACAM5) were evaluated in migration, invasion, and adhesion assays in vitro using a panel of human pancreatic, breast, and colonic cancer cell lines, and in the GW-39 human colonic micrometastasis model in vivo. MN-3 Fab' and MN-15 Fab' were both effective at inhibiting cell migration. MN-15 Fab' treatment inhibited invasion, reducing cell penetration through an extracellular matrix (ECM). MN-3 Fab' also decreased invasion but was less effective than MN-15 Fab' in four of five cell lines. All three monoclonal antibody (mAb) Fabs decreased adhesion of tumor cells to endothelial cells by 49% to 58%. MN-15 Fab' but not MN-3 or MN-14 Fabs induced a decrease in adhesion of three of six cell lines to the ECM protein, fibronectin, but adhesion to vitronectin, laminin, collagen-I, and collagen-IV was not affected. In vivo studies showed that treatment with MN-3 Fab' or MN-15 Fab' of mice implanted with GW-39 human colonic cancer cells increased their survival (P < 0.025 and P < 0.01, respectively). These studies show that antibody Fabs that target either CEACAM5 or CEACAM6 affect cell migration, cell invasion, and cell adhesion in vitro, and that MN-15 and MN-3 Fabs have antimetastatic effects in vivo, resulting in improved survival of mice with metastases. Thus, blocking the N and A1B1 domains of CEACAM5/CEACAM6 can impede the metastatic process.
|New functions for the matrix metalloproteinases in cancer progression. |
Egeblad, Mikala and Werb, Zena
Nat. Rev. Cancer, 2: 161-74 (2002) 2002
Matrix metalloproteinases (MMPs) have long been associated with cancer-cell invasion and metastasis. This provided the rationale for clinical trials of MMP inhibitors, unfortunately with disappointing results. We now know, however, that the MMPs have functions other than promotion of invasion, have substrates other than components of the extracellular matrix, and that they function before invasion in the development of cancer. With this knowledge in hand, can we rethink the use of MMP inhibitors in the clinic?
|A new in vitro assay for quantitating tumor cell invasion. |
Repesh, L A
Invasion Metastasis, 9: 192-208 (1989) 1989
The attachment to and penetration of basement membranes by tumor cells is required to complete the metastatic cascade which culminates in the establishment of secondary tumor foci. Therefore, basement membranes are critical barriers to the passage of disseminating tumor cells. We have developed a simple, in vitro model using matrigel-coated transwell chambers (Costar) for use in a tumor cell invasion assay. Two variants of the K1735 UV-induced murine melanoma cell line were assayed for their invasive capabilities and compared with their ability to colonize the lung in an experimental metastasis assay. The K1735-M2 cells, which are highly metastatic in vivo, invaded through basement membrane matrigel at a significantly higher rate than the low metastatic cells, K1735-16, in a 72-hour assay. As a negative control, normal murine fibroblasts were incapable of penetrating the barrier. Tumor cell invasion in vitro correlated with lung colonization in vivo. Therefore, this model may provide a valuable tool to study the mechanisms involved in the pathogenesis of tumor cell invasion during hematogenous dissemination.
|A rapid in vitro assay for quantitating the invasive potential of tumor cells. |
Albini, A, et al.
Cancer Res., 47: 3239-45 (1987) 1987
We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.
|Use of a reconstituted basement membrane to measure cell invasiveness and select for highly invasive tumor cells. |
Terranova, V P, et al.
Proc. Natl. Acad. Sci. U.S.A., 83: 465-9 (1986) 1986
Malignant cells must traverse basement membranes during their migration to sites distant from the primary tumor. Since basement membranes are thought to be a critical barrier to the passage of tumor cells, we have constructed a model basement membrane-stromal matrix consisting of laminin and type IV collagen reconstituted onto a disk of type I collagen for use in an in vitro assay of invasiveness. Metastatic tumor cells and leukocytes are able to cross this barrier, whereas nonmetastatic tumor cells, fibroblasts, and epidermal cells cannot penetrate it. Those tumor cells that penetrate the barriers were found, when isolated and subcultured, to be more invasive and to produce more metastases than the parental population. This assay system should be useful for studying the invasiveness of tumor cells and for isolating highly invasive variants.
|Tumor invasion and metastases: role of the basement membrane. Warner-Lambert Parke-Davis Award lecture. |
Liotta, L A
Am. J. Pathol., 117: 339-48 (1984) 1984
|Advancing cancer research: From hallmarks & biomarkers to tumor microenvironment progression|
|Cell Migration and Invasion: Choosing the Right Assay|
|QCM Collagen Cell Invasion Assay, Colorimetric - Data Sheet|
|QCM Collagen Cell Invasion Assay, Colorimetric - Data Sheet|