LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro
© Ghislin et al.; licensee BioMed Central Ltd. 2012
Received: 23 January 2012
Accepted: 1 October 2012
Published: 5 October 2012
Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration.
A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software.
We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration.
Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.
KeywordsMelanoma Transendothelial migration Metastasis LFA-1 ICAM-1 HUVEC
Metastatic melanoma account for most skin cancer deaths. When diagnosed early, primary, non-disseminated, tumors are successfully eliminated through excision. However, in about 20% of the cases, dissemination of tumor cells leads to aggressive forms of cancers highly refractory to chemotherapy, with a median survival rate of 6 months . Uncovering molecules required for melanoma metastasis is therefore essential.
Hematogenous metastasis of cancer consists of several steps enabling cancer cells to intravasate, to survive in the blood circulation and to adhere to the vessels, eventually extravasating and establishing new metastatic lesions. Extravasation of most cancer cells largely mimics leukocyte transendothelial migration from the blood flow into sites of tissue inflammation . This controlled process involves the multistep action of traffic signals and adhesion molecules that mediate rolling, adhesion and transendothelial migration of lymphocytes .
The role of cell adhesion molecules (CAMs), such as intercellular cell adhesion molecule-1 (ICAM-1), vascular endothelial cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin, has been studied extensively in the process of inflammation . Indeed, leukocyte adhesion during inflammation is thought to proceed in a cascade-like fashion, in which selectins are responsible for leukocyte capture and rolling, and integrins for mediating firm adhesion and transmigration [5, 6]. Among these integrins, Leukocyte Function-Associated antigen-1 (LFA-1; αLβ2) composed of two chains, CD11a and CD18, has been extensively described for its essential role in leukocyte extravasation [2, 7]. It functions as a receptor for ICAM-1 (CD54) [8–10]. Besides its role in the firm adhesion of leukocytes to the endothelium, it appears dominant in transendothelial migration [7, 11]. In addition numerous studies have shown that complete inhibition of CD18, or genetic mutations in CD18 profoundly reduce leukocyte transmigration at sites of inflammation .
Junctional Adhesion Molecules A (JAM-A) can also interact with LFA-1 via its second membrane-proximal Ig domain [13, 14]. During leukocyte transendothelial migration, the homophilic transendothelial interactions between two molecules of JAM-A must be disrupted to enable a migrating leukocyte to pass through junctions  and it has been evidenced that LFA-1 binding to JAM-A destabilizes the JAM-A homophilic interaction, thus allowing transendothelial migration to proceed .
LFA-1 has been studied in different tumors, for instance myelomas and gastrointestinal carcinomas. It has been shown that expression of LFA-1 correlates with the aggressiveness of myeloma  and is present in metastatic gastrointestinal carcinomas .
In melanoma cell lines, LFA-1 cell-surface expression is not detected. Towards a molecular explanation to the high capacity of melanoma tumor cells to metastase, two groups proposed that melanoma cells interact with neutrophils, thereby suggesting that neutrophils might be used as carriers by the tumor cells [19–21]. Liang et al. have notably demonstrated that under IL-8 signaling, melanoma interact with polymorphonuclear neutrophils (PMNs) through the binding between ICAM-1 on melanoma cells and β2 integrins on PMNs. The authors also showed that this interaction facilitates melanoma cell adhesion to the endothelial cells and subsequent extravasation by a shear-rate dependent mechanism .
However during our studies of melanoma metastasis, we observed that melanoma cell lines have the capacity to transmigrate through endothelial monolayers in the absence of PMNs. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the ICAM-1/LFA-1 ligand-receptor interaction. In this manuscript we studied three human melanoma cell lines with differential transmigration capacities. We provide evidence that melanoma supernatants induce ICAM-1 expression on HUVEC cells, and that LFA-1 can be detected on melanoma cell lines when using HUVEC-conditioned medium. Further confirmation was obtained through the use of either ICAM-1 or LFA-1 blocking antibodies introduced during the co-culture and show that they strongly impair melanoma transmigration.
Material and methods
Cell lines and cell culture
The melanoma cell line SLM8 kindly provided by M. Viguier (Service de dermatologie, Hopital Saint-Louis, Paris, France), is derived from a lymph node metastasis. The 1205LU cell line, a generous gift by A. Mauviel (Institut Curie/CNRS UMR 3347/Inserm U1021), is derived from the lung metastatic WM793 cell line. The A375 cell line was purchased from the ATCC. HUVEC cells were kindly provided by C. Nahmias (Institut Cochin, Paris, France). A375, 1205LU and SLM8 human cell lines were grown in DMEM/F12 (Invitrogen, Cergy-Pontoise, France) supplemented with antibiotics and 10% fetal calf serum (FCS) in a 5% CO2 atmosphere (hereafter named FCS complete medium). HUVEC cells have been cultured in high glucose DMEM containing glutamax, 1% antibiotics and 10% FCS in a 5% CO2 atmosphere.
To obtain conditioned medium cells were grown to confluence during 48hrs before collecting the supernatant. The medium was diluted to 3/4 with FCS complete medium (1/4) then added for 24hrs to the target cells plated at 80% of confluence. The basal negative control was obtained with only FCS complete medium.
RNA extraction and semi-quantitative PCR
Primer sequences and PCR settings
Number of cycle
Cells (5 × 105) treated with conditioned medium from a 48hrs of HUVEC cells culture were harvested in cold phosphate-buffered saline (PBS) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and incubated for 20 min with 2% fetal calf serum in PBS. Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml. After three washes, cell death was monitored by propidium iodide (5 μg ml) uptake and fluorescence was analyzed in a cytometer from DAKO (Trappes, France).
Trans-endothelial migration of melanoma
Trans-endothelial migration of melanoma was performed as previously described . Briefly, 5 × 104 HUVEC cells were seeded on 2mg/ml type I collagen-coated Transwell culture inserts with 8 μm pores (Greiner Bio-One SAS, Courtaboeuf, France) and grown for 2 days. Melanoma cells were labeled with 10 μM of the lipophilic fluorescent dye DiO (Molecular Probes, Invitrogen, Cergy-Pontoise, France) for 20 minutes at 37°C. 5×104 fluorescent melanoma cells were added to wells containing either 1μg/ml of isotypic control (IgG from BD Pharmingen, San Diego, CA, USA); or anti-CD11a (ab3981; Abcam; Paris, France), anti-CD18 (ab8220; Abcam; Paris, France) or anti-ICAM-1 (MAB2146Z; Millipore; Molsheim France) antibodies in the upper chamber. A chemotactic gradient was created by addition of 10% FCS to the lower chamber. Melanoma cells were allowed to migrate at 37°C and 5% CO2 for 48 hours.
To remove non-migrating cells, the ones on the upper face of the filter were gently scraped using a cotton swab. Cells on the lower face were washed in PBS, fixed with 4% formaldehyde for 10 min and washed in PBS. Nuclei were then labeled with 1μg/ml DAPI for 5min and cells were washed again. The migrating melanoma cells were observed under an epifluorescence microscope using a magnification of x100. Three to five pictures of adjacent fields of the central zone of each Transwell were taken. Fluorescence intensity was quantified with the ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Histograms display the data obtained with 3 independent experiments, and all the experiments were performed in duplicate. The p value has been calculated by an ANOVA statistical test.
Human cytokine array
The detection of cytokines’ production by the three melanoma cell lines has been tested after 24hrs of culture in 12ml of culture medium using the Proteome Profiler™ Array Human Cytokine Array Panel A according to the manufacturer’s instructions (ARY005; R&D systems Minneapolis, USA). Hence, melanoma cells have been tested on nitrocellulose membranes each containing 36 different anti-cytokine antibodies spotted in duplicate. Experiments have been done in duplicate.
Melanoma cells transmigrate through endothelial cells
Transwell experiments mimicking transendothelial migration were performed with three melanoma cell lines. Briefly, HUVEC cells (Human Umbilical Vein Endothelial Cells) are grown as a monolayer to confluence on a type I collagen matrix layer poured onto a microporous membrane. Melanoma cells, stained with a lipophilic fluorescent dye, are then allowed to cross this barrier for 48hrs. The corresponding membranes of the Transwells are photographed prior to the quantification of fluorescent cancer cells, located at the lower part of the membrane.
These data indicate that melanoma cells have the capacity in vitro to cross endothelial cell monolayer, although PMNs are not present. We therefore studied the molecular mechanisms leading to this phenomenon.
LFA-1 is involved in melanoma trans-endothelial migration in vitro
Melanoma cell lines enhance the expression of ICAM-1 on the HUVEC cells
As exogenous inflammation molecules are not used in the transmigration assays displayed in this report, we wondered if melanoma cell lines could induce ICAM-1 expression in HUVEC cells. To answer this question, conditioned medium was prepared from the melanoma cell lines after 48hrs of culture. The HUVEC cell line was next cultured with this conditioned medium and ICAM-1 transcript expression was analyzed. Figure 4B shows that ICAM-1 is up-regulated by the conditioned medium originating from all three melanoma cell lines, more efficiently with the A375 and 1205LU cell supernatants.
We therefore conclude that preferentially the A375 and 1205LU melanoma cell lines can transmigrate through the endothelial cells due to a binding between ICAM-1 expressed by the HUVEC cells and LFA-1expressed by tumor cells during their co-culture.
LFA-1 is involved in clumps’ formation
The recruitment of lymphocytes to sites of inflammation involves a sequence of rolling along capillary vessel walls, followed by chemokine induced arrest and migration across a tight layer of vascular endothelial cells [35, 36]. Invasion of tumor cells to secondary sites is often compared with lymphocyte transendothelial migration since several common cell adhesion molecules, such as selectins and integrins, are involved in both lymphocyte and tumor cell migration. It has previously been shown that following secretion of IL-8 in the tumor microenvironment, the PMN facilitated melanoma extravasation via the binding of β2 integrins on PMNs and ICAM-1 on melanoma cells . In a congress report, it was mentioned (although not confirmed through a publication) that the co-culture of HUVEC cells and a melanoma cell line from a lymph node metastasis promotes the expression of αVβ3 facilitating melanoma extravasation . In this report, we focused on the direct role of a major integrin, LFA-1 and its counter receptor ICAM-1 in melanoma transmigration. To investigate this hypothesis we used three melanoma cell lines displaying different efficiencies of transendothelial migration but similar expression of the tested integrin. With blocking antibodies against these integrins and ICAM-1, we showed that LFA-1 promotes melanoma cells transmigration in vitro. In addition we provide evidence that LFA-1 seems to be responsible for clumps’ formation, which could enhance further their extravasation capacities. This is consistent with previous reports examining the migration of dendritic cells  or extravasation of some tumors [40, 41].
Patients with metastatic melanoma have a reduced life span in the range of 6 months. Limited therapeutic treatments explain this poor prognosis. Our findings showing that CD18 blockade impairs melanoma cell transmigration might provide a new tool to control melanoma metastatic mechanism.
Competing of interest
The authors’ declare that they have no competing interests.
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