Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal tract. Small intestinal GISTs appear to be associated with poorer prognosis and higher metastasis rate than gastric GISTs of the same size and mitotic index. Recently, we reported that cell adhesion molecule 1 (
Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal (GI) tract (
GISTs are neoplasms with malignant potential varying from virtual indolence to rapid progression. Up to 20% of GIST patients have overt metastases at diagnosis, and the metastases typically occur in the abdominal cavity or the liver. Small intestinal GISTs are considered to have a worse prognosis than gastric GISTs because of their higher risk of metastasis and tumor-related death (
We have recently reported that cell adhesion molecule 1 (
In the present study, we examined whether CADM1 affects proliferation, migration, invasion, adhesion to endothelial cells, and transendothelial migration of GIST cells. GIST-T1 cells with high
Fresh tissue samples of a representative gastric GIST and a representative small intestinal GIST were collected intraoperatively, frozen, and stored at −80°C until use. RNA and protein were extracted from the samples. The RNA and protein in the present experiments have been used in previous experiments (
A human GIST cell line, GIST-T1, which is derived from a metastatic pleural tumor from gastric GIST in a Japanese woman, was purchased from Cosmo Bio. It harbors a heterozygous c-
Full length of
GIST-T1 cells, GIST-T1-CAD cells, a representative gastric GIST tissue, and a representative small intestinal GIST tissue were lysed in CelLytic M Cell Lysis Reagent (Sigma-Aldrich; Merck KGaA) containing 5 mM NAF, 1 mM Na3VO4, and proteinase inhibitor cocktail (Roche). As described previously (
Total RNA was extracted from GIST-T1 cells, GIST-T1-CAD cells, a representative gastric GIST tissue, and a representative small intestinal GIST tissue using RNeasy mini kit (Qiagen, Inc.), and 10 µg of total RNA was applied for RT-qPCR templates. As described previously (
GIST-T1 and GIST-T1-CAD cells were plated in 24-well plates (Corning Incorporated) at 2×104 cells per well in growth medium. After incubation for 1, 3, 5, and 7 days, cells were trypsinized, resuspended in Accumax (Innovative Cell Technologies), and counted by hemocytometer (Z1, Beckman Coulter). Six wells were used for each cell type in each experiment. The cell proliferation assay was repeated three times.
Migration ability was assessed by the wound-healing assay. GIST-T1 and GIST-T1-CAD cells were seeded in 6-well plates at 3×105 cells and allowed to grow to 90% confluence. The cell monolayer was scratched with a sterile micropipette tip, and then serum-free medium was added into plates after washing the cells thrice with PBS. Photographs of images captured at ×200 magnification were taken at the same six selected locations for each well under a phase contrast microscope (All-in-One Microscope; Keyence). The area that remained clear after 0, 1, 2, 3, and 4 days was quantified with ImageJ (National Institutes of Health) and the covered area was calculated by comparing to the area of the wound at day 0. This assay was repeated three times.
The migration assay was performed using Falcon cell culture inserts (Corning Incorporated) without Matrigel and the invasion assay was performed using 24-well BD Bio-Coat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer's protocol. GIST-T1 and GIST-T1-CAD cells were resuspended at a density of 5×105 cells/l in 0.5 ml of the serum-free medium and added into the upper chamber of the insert. DMEM supplemented with 10% FBS (0.75 ml) was added to the lower chambers. After incubation for 2 days, non-migrated or non-invaded cells were removed from the upper surface of the membranes using a cotton tipped swab. The cells adhering to the bottom surface of the membrane were fixed and permeabilized in 10% neutral formalin and 100% methanol, respectively. Migrated or invaded cells were stained by Giemsa staining and counted in nine selected microscope fields per membrane. The experiments were conducted three times.
Static adhesion assay using fluorescence-labeled tumor cells was performed. HUVECs (2.5×105 cells/well) pretreated with or without 10 ng/ml TNF-α (Invitrogen; Thermo Fisher Scientific) were cultured in 96-well plates overnight. TNF-α has the potential to stimulate endothelial cell adhesion. GIST-T1 and GIST-T1-CAD cells were labeled with 2 µg/ml Calcein-AM (Dōjindo Laboratories) at 37°C for 30 min, washed thrice with PBS, and resuspended at 2.5×106 cells/ml with serum-free DMEM, and followed by pipetting onto confluent HUVECs monolayers. After coculturing for 2 h, medium and unbounded tumor cells were removed and discarded. Adherent tumor cells and endothelial cells were washed three times with PBS. Then the amount of Calcein-AM fluorescence was measured using a fluorescence microplate reader (2030 ARVO X4, PerkinElmer Life and Analytical Sciences), at an excitation wavelength of 485 nm and emission wavelength of 530 nm.
HUVECs (2×105) pretreated with 10 ng/ml TNF-α were seeded onto 24-well Transwell Inserts and cultured overnight. After formation of a confluent HUVEC monolayer, tumor cells labeled with Calcien-AM were added to the upper chamber, and cells were cocultured for 48 h. After incubation, the non-migrated cells which were present on the upper side of the membrane were removed with a cotton tipped swab, and the transmigrated cells on the bottom side of the membrane were fixed with 10% neutral formalin. Transmigrated cells were visualized using a fluorescence microscope and counted from 10 random fields under ×200 magnification. Experiments were performed in triplicate and repeated three times.
Statistical analysis of proliferation assay, wound-healing assay was performed by two-way mixed ANOVA followed by Bonferroni's multiple comparison test. The significance of cell migration and Matrigel invasion in transwell assay, adhesion assay and transendothelial migration assay was analyzed by unpaired Student t-test. P<0.01 was considered statistically significant.
GIST-T1 cells are a cell line of GIST cells originally derived from the stomach. Consistent with our previous report (
To investigate whether CADM1 is involved in the proliferation of GIST cells, we compared proliferative ability between GIST-T1 cells and GIST-T1-CAD cells. Cell number was counted at days 0, 1, 3, 5, and 7 after seeding with 2×104 of both cells. After day 3, the number of GIST-T1-CAD cells was significantly smaller than the number of GIST-T1 cells (
To explore the effect of CADM1 on GIST cell-vascular endothelial cell adhesion, we performed a static adhesion assay based on the binding of fluorescence-labeled GIST-T1 cells and fluorescence-labeled GIST-T1-CAD cells to HUVECs monolayers. We firstly examined the ability of tumor cells to adhere to TNF-α-unstimulated endothelium. GIST-T1-CAD cell-HUVEC adherence was 16.6 times greater than GIST-T1 cell-HUVEC adherence (
We have recently reported that
Expression of
In the present study, expression of
Recently, anti-CADM1 antibodies were developed as a promising candidate agent for reducing ATLL cell invasion via blocking cell adhesion. The antibodies appear to show a minimal cytotoxic effect on the growth of the ATLL cell line (
There are some limitations in our study. First, we did not examine expression of surface adhesion molecules other than
In summary, CADM1 in GISTs might act as a suppressor of tumor growth, migration, and matrix invasion, but stronger GIST cell-endothelial cell interaction induced by high
Not applicable.
All data generated or analyzed during this study are included in this published article.
JY, TK, NK, TY, MY, KI, AI and SH participated in data collection and discussion of the findings. JY, TK, NK, TY and MY carried out the experiments. JY and TK performed the statistical analysis. JY and SH wrote the manuscript. JY and SH confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Experiments using recombinant DNA were approved by the Committee for Recombinant DNA Experiments of Hyogo College of Medicine (approval no. 24015; Nishinomiya, Japan). The use of fresh human gastrointestinal stromal tumor (GIST) tissue samples for GIST assays, including gene analysis, was approved by the Ethical Committee of Hyogo College of Medicine (approval no. 28), and the patients/participants provided their written informed consent to participate in this study.
Not applicable.
The authors declare that they have no competing interests.
GIST-T1-CAD cells show high expression of CADM1. (A) The relative protein level of CADM1 in GIST-T1-CAD and GIST-T1 cells, and s-GIST and g-GIST tissues was examined by western blotting. (B) Level of