A comparative evaluation of various invasion assays testing colon carcinoma cell lines

Abstract

Summary Various colon carcinoma cell lines were tested in different invasion assays, i.e. invasion into Matrigel, into confluent fibroblast layers and into chicken heart tissue. Furthermore, invasive capacity and metastatic potential were determined in nude mice. The colon carcinoma cells used were the human cell lines Caco-2, SW-480, SW-620 and HT-29, and the murine lines . None of the human colon carcinoma cells migrated through porous membranes coated with Matrigel; of the murine lines, only Colon-26 did. When incubated in a mixture of Matrigel and culture medium non-invading cells formed spheroid cultures, whereas invading cells showed a stellate outgrowth. Only the heterogeneously shaped (epithelioid and stellate) cells of SW-480 and SW-620 and the spindle-shaped cells of Colon-26 invaded clearly confluent skin and colon fibroblasts as well as chicken heart tissue. However, when transplanted into the caecum of nude and syngeneic mice, all the lines tested were invasive with the exception of Caco-2 cells. We conclude that the outcome of in vitro tests measuring the invasive capacity of neoplastic cells is largely dependent on the test system used. Invasive capacity in vitro is strongly correlated with cells having a spindle cell shape, vimentin expression and E-cadherin down regulation. In contrast, HT-29 and Colon-38 cells having an epithelioid phenotype were clearly invasive and metastatic in vivo, but not in vitro. © 1999 Cancer Research Campaign Keywords: Matrigel; confluent fibroblast layers; chicken hearts; orthotopic transplantation 934British Journal of Cancer (1999) 81(6), 934-941 © 1999 Cancer Research Campaign Article no. bjoc.1999 Received 2 In vitro testing Matrigel assayThe assay was carried out as described by Albini et al (1987). Perforated polyvinylpyrrolidone-free polycarbonate membranes of 13-mm diameter (Nucleopore, CA, USA) were used with 8-µm pore size, which is sufficiently large to allow the passage of single carcinoma cells.To promote attachment all the membranes were first coated on the lower side in 4-well dishes with 15 µl of a 0.1% solution of fibronectin (Sigma, St Louis, MO, USA) in serum-free medium according to Sieuwerts et al (1997). Subsequently half of the membranes were coated with a layer of Matrigel to measure invasion. An amount of 20 µl containing 40 µg Matrigel was applied per membrane surface obtained by dilution of a stock solution with serum-free medium as recommended (Collaborative Biomedical Products, Becton and Dickinson Labware, Bedford, UK). The concentration used prevents preliminary detachment of the Matrigel from the filters and falls within the range of concentrations used by others (see Discussion). The Matrigel was gelatinized at 37°C for 20 min. The other half of the membranes was left uncoated to measure migration.The membranes were sealed in a sterilized Boyden chemotaxis chamber (Nucleopore Membrane Products, CA, USA) after filling the lower compartment with 220 µl of serum-free NIH-3T3 conditioned medium as attractant. The upper compartment was ...

Influence of the microenvironment on invasiveness of human bladder carcinoma cell lines

“…The SD cell line was not invasive in vitro, but after subcutaneous injection and after inoculation in the bladder wall, SD cells developed muscle-invasive tumors. Similar discrepancies between the embryonic chicken heart invasion assay and in vivo invasiveness were reported earlier in a study on colorectal carcinoma cell lines [11,12]. Our results support the view that the microenvironment may regulate invasive behavior of bladder carcinoma cells in vivo, for instance by influencing the expression of E-cadherin, as suggested by Mareel et al [21] or by induction of synthesis of extracellular matrix degrading proteins [33].…”

Tead and AP1 Coordinate Transcription and Motility

“…Furthermore, we found that Crispr-mediated SRC1/2/3 or Dock4/9 knockout inhibited YAP5SA/JunD-dependent migration in HT29 cells (Figure 5H–J), highlighting the functional importance of these proteins in YAP/Tead-AP1 cooperation. Next, we demonstrated that the combined inhibition of Tead and AP1 activity in the highly invasive HCT116 colon cancer cells (de Both et al, 1999) led to effective inhibition of cell migration and invasion, measured by cell scratch and matrigel invasion assays (Figure 5K–M). Moreover, we analyzed the expression of JunD and Tead4 in sixty two human lung adenocarcinomas and their matched lymph node metastases, and found that JunD and Tead4 proteins were expressed significantly higher in the lymph node metastases than in the matched primary cancers (Figure 5N–Q), suggesting that the potential involvement of Tead-AP1 cooperation in promoting tumor invasion and metastasis.…”

“…To test this hypothesis, we ectopically expressed YAP5SA and JunD in the HT29 cells, a relatively less invasive colon cancer cell line (de Both et al, 1999), and found that YAP5SA and JunD co-expression significantly promoted migration of HT29 cells, measured by transwell migration assay (Figure 5F). In addition, we showed that YAP5SA or TAZ4SA (an active form of TAZ) induced migration was partially inhibited by DN-JunD (Figure 5G).…”

Activation of Human Meprin-α in a Cell Culture Model of Colorectal Cancer Is Triggered by the Plasminogen-activating System

“…Invasion and migration assays based on the already established co-culture model will clarify this issue in future experiments. Although Caco-2 cells appear to be noninvasive in nude mice (65), secretory products of cells in the vicinity may interact at the surface of the cancer cells and by this increase their invasive potential. For instance, migration of Caco-2 cells in wounded cell layers was stimulated 2-3-fold by exogenous u-PA, an effect dependent on binding to u-PAR (57).…”

“…In vitro, trypsin cleaves the propeptide of human meprin following an Arg residue (Arg 65 in human meprin-␣ and Arg 61 in human meprin-␤) (18), and this is most probably the physiologic mechanism of promeprin activation in the intestinal lumen. However, possible other meprin-activating proteases must exist in vivo, especially in meprin-expressing tissues or cells, where trypsin is absent.…”