Human colon carcinoma (HCT-8) cells show metastatic phenotype when cultured on appropriately soft substrates. Here, we studied the surface non-specific adhesion in HCT-8 cells throughout the in vitro metastasis process. A novel bio-MEMS force sensor was used to measure the cell-probe non-specific adhesion. The adhesion characteristics are analyzed using classical Johnson-Kendall-Roberts (JKR) theory. Our results indicate that the post-metastatic HCT-8 cells (dissociated R cells) display remarkably diminished surface adhesion and are potentially more invasive than original pre-metastatic HCT-8 cells (E cells). To the best of our knowledge, this is the first quantitative data on cancer cells adhesion change as in vitro metastasis proceeds.
It is well known that, during in vivo cancer metastasis, malignant cancer cells reduce their surface adhesion (both specific and non-specific) [1] as well as modify their extracellular matrix (ECM) ligands [2] to detach from primary tumor and enhance successful invasion into distant healthy organs. Simultaneously, cancer cells down-regulate their surface cell-cell adhesion molecules, i.e. E-Cadherin, to escape from tumor and initiate metastasis [1]. However, there is no quantitative report on cancer cell adhesion throughout the entire metastasis process, since in vivo metastasis is nearly impossible to detect [3].
We had discovered [4] that human colon cancer cells (HCT-8) can consistently display an in vitro metastasis-like phenotype (MLP) within only 7 days of culture on soft hydrogel substrates with appropriate mechanical stiffness (Poly-acrylamide gels with Elastic modulus: 21∼ 47 kPa [14, 15]). We found that MLP is consistent, repeatable and irreversible (Fig. 1a-1c). In addition, the post MLP cancer cells (referred to here as R cells meaning round-shaped in contrast to the E-cells, i.e., the original HCT-8 cells that are epithelial in nature) up-regulate a number of in vivo tissue-destructive proteinases, such as, MMPs [4]. R cells also express remarkably diminished E-Cadherin patterns compared to HCT8 E cells (Fig. 1d, 1e). Using this model system, we are able to study the kinetics of non-specific and specific surface adhesion change on HCT-8 cancer cells.
In this paper, we measure the non-specific adhesion of both pre and post metastatic HCT-8 cells (E and R cells respectively) using a novel bio-MEMS force sensor. The adhesion energy and other mechanical properties are analyzed using classical Johnson-Kendall-Roberts (JKR) theory [5]. We find that after undergoing metastasis (or MLP), the dissociated HCT-8 cells (R cells) down-regulate non-specific adhesion, in contrast to their ancestors, HCT-8 E cells. The reduction of non-specific adhesion is coincident with the immuno-fluorescent staining data of cell-cell specific adhesion molecule E-Cadherin, which shows 4 ∼ 6 times down-regulation after MLP (Fig. 1d-1e).
The bio-MEMS sensor consists of a micro cantilever beam with spring constant k = 3.48 nN/ μm. A flat probe is attached with the beam which forms adhesive contact with cells. The sensor is made from single crystal silicon, and is coated with a thin layer of native silicon oxide (SiO2). The probe and the sensor are not functionalized. The sensor is manipulated with an x-y-z piezo stage.
To measure the cell adhesion, the flat probe is brought in contact with cells’ lateral convex surface at the boundary. After a 2-minute contact, force sensor is pulled away horizontally from the cell island at a constant quasi-static speed of 2.1 ± 0.4 μm/s (Fig. 2a). Due to the cell-probe adhesion, the sensor beam deforms during retraction. Corresponding restoring force of the cell island is given by F = kδ (Fig. 2a-c). Note the probe is non-functionalized (free of any extra-cellular matrix proteins), and only has a coating of SiO2 on the surface due to air exposure.
During probe retraction, the cell is continuously stretched while the cell-probe contact area radius Rc remains unchanged (Fig. 3b-e) and the contact angle θ increases (Fig. 3b). At critical value of force, Fc, the cell suddenly detaches from probe (Fig. 3d). The critical Fc at detachment is optically recorded by video camera and was determined as 27.8 ± 2.2 nN.
A similar experiment on cells after MLP shows so measurable adhesion, i.e, the force to detach was zero for all the cells tested. Figure shows the measured adhesion in pre and post metastatic cells.
Levels of Soluble E-Cadherin in Breast, Gastric, and Colorectal Cancers