Cell Invasion / Extravasation

The ECIS Method

We have used the ability of ECIS to detect changes in cell morphology to design whole-cell assays related to the behavior of the cancer cell including metastatic potential.

The measurement is based upon observations of transformed cells interacting with established normal cell layers. Several years ago Kramer and Nicolson presented scanning electron micrographs demonstrating, in cell culture, the metastatic-like behavior of cancer cells. In this work they first established a confluent layer of endothelial cells in culture. This cell layer was then challenged with highly metastatic cells (in this case melanoma B16 cells). Electron micrographs taken at different time intervals showed the melanoma cell attaching to the endothelial cell layer, with subsequent retraction of the endothelial cells, and finally penetration of the melanoma to the tissue culture substratum.

Since the ECIS system should easily detect this alteration in the endothelial cell layer, we have explored an assay to monitor these changes to look for correlations of impedance changes with the metastatic potential of cancer cells.

Our studies were done in collaboration with researchers in Dr. Donald Coffey's group at Johns Hopkins Brady Urological Institute in Baltimore, MD. Workers there had previously established a series of cancer cell sublines. These all originated from a culture established from a single rat prostatic tumor, that, upon extensive tissue culture passages and mutagenesis, resulted in several sublines that exhibited a wide range of metastatic potential. When injected subcutaneously into rats, some of these sublines formed tumors only at the site of injection (G subline). Others would form distant metastases approximately 10 to 20% of the time (AT1, ST3 sublines); whereas, others would formed distant metastases at several locations over 90% of the time (AT3, MLL sublines). The basic plan of our experiments was to first establish a confluent layer of endothelial cells and then follow impedance changes as the layer was exposed to different rat prostatic sublines.

Establishment of the endothelial cell layer

These ECIS data show the establishment of the endothelial cells layer for the assay. The ECIS wells were first coated with an adsorbed layer of gelatin (1.0 mg/ml gelatin in 0.15M NaCl applied for 5 minutes and then washed out of the well with sterile medium). At time zero, a HUVEC (human umbilical vein endothelial cell) suspension was added to several wells and their attachment and spreading monitored with standard ECIS measurements. The resistive portion of the impedance at 4000 Hz is shown as a function of time. 100,000 cells were added to each well in complete medium (DMEM with 10% FBS). Spreading is completed in the first 2 hours after inoculation and the subsequent fluctuations are characteristic of this cell and are related to cellular motion.
Challenge of the HUVEC layer

The drawing shows the type of activities expected to occur during the challenge of the normal endothelial cell layer with the metastatic cell lines.
ECIS data showing control culture receiving additional medium

The data shows a control culture receiving only additional medium. The other traces are of duplicated cultures challenged with the weakly metastatic G subline or the metastatic AT3 subline. All additions were at the two vertical marks on the x-axis. Note that the addition of both cell sublines tends to reduce the resistance of the system and result in reduction of the fluctuations. The activity of the AT3 subline is considerably larger than that of its weakly metastatic counterpart (G). The loss of the resistance is due to the lost of integrity of the endothelial cell layer in response to the activities of the cancer cells.
Challenge with active and heat-killed cells

These curves show similar activity where the highly metastatic MLL cell line rapidly breaks down the resistance of the established endothelial cell layer, but the same cells when first heat killed (15 minutes at 56 degrees C) have no effect verifying that the assay is indeed seeing biological activities. In these data all additions are at time zero. challenge of HUVEC cell layers

ECIS Measurements of Metastatic Potential
Related ECIS Publications

Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis. M Tarantola, AK Marel, E Sunnick, H Adam, J Wegener, and A Janshoff. Integr Biol (Camb). 2010; 2: 139. 

ALCAM, Activated Leukocyte Cell Adhesion Molecule, Influences the Aggressive Nature of Breast Cancer Cells, a Potential Connection to Bone Metastasis. SIMON DAVIES and WEN G. JIANG. Anticancer Res. 2010; 30:1163-1168. 

Chen, J., Ye, L., Zhang, L., Jiang, W.G., “Placenta Growth Factor, PLGF, Influences the Motility of Lung Cancer Cells, the Role of Rho Associated Kinase, Rock.” J of Cellular Biochemistry: 105:313-320 (2008).

Saxena, N.K., Sharma, D., Ding, X. Lin, S., Marra, F., Merline, D. Anania, F., “Concomitant Activation of the JAK/STAT, P13K/AKT and ERK Signaling is Involved in Leptin-Mediated Promotion of Invasion and Migration of Hepatocellular Carcinoma Cells.” Cancer Research: 2497-2507 (2007).

Ren, J., Xiao, Y., Singh, L.S., Zhao, X., Zhao, Z., Feng, L., Rose, T.M., Prestwich, G.D., Xu, Y., “Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion, Migration, and Invasion of Ovarian Cancer Cells.” [PDF] Cancer Res 2006; 66: (6) (2006).

Wang, H.S., Hung, Y., Peng, S.T., Guo, Y.J., Lai, M.C., Liu, C.Y., Hsu, J.W., “CD44 Cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (æLß2) and VLA-4 (æ4ß1).” Experimental Cell Research 304: 116-126 (2005).

Sawhney RS, Sharma B, Humphrey LE, Brattain MG. “Integrin alpha2 and extracellular signal-regulated kinase are functionally linked in highly malignant autocrine transforming growth factor-alpha-driven colon cancer cells.” J Biol Chem. 278(22):19861-9 (2003).

Sriram, P.S., Mohammed, K.A., Nasreen, N., Hardwick, J., Van Horn, R., Sanders, K., Antony, V.B. “Adherence of ovarian cancer cells induces pleural mesothelial cell (PMC) permeability.” Oncol Res. 13(2):79-85 (2002).

Keese, C. R. Bhawe, K. Wegener, J. Giaever, I. “Real-Time Impedance Assay to Follow the Invasive Activities of Metastatic Cells in Culture,” BioTechniques 33:842-850 (October 2002).