A role for heterologous gap junctions between melanoma and endothelial cells in metastasis

F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found that connexin (Cx) 26 is upregulated in BL6 cells. To examine gap junction formation, we devised a coculture system, in which an opened vein segment was placed at the bottom of a culture dish and then dye-labeled melanoma cells were seeded onto it. Immunohistochemistry indicated that the vein segment preserved the integrity of the endothelial monolayer. In this system, BL6 cells could transfer dye into endothelial cells but F10 cells could not. Transfection with wild-type Cx26 rendered F10 cells competent for coupling with endothelial cells and as spontaneously metastatic as BL6 cells. Conversely, transfection with a dominant-negative form of Cx26 rendered BL6 cells deficient in coupling and less metastatic. In human melanoma lesions, the level of Cx26 expression was low in melanoma cells residing in the basal layer, but significantly upregulated in melanoma cells invading the dermis. The results suggested that Cx26 plays a role in intravasation and extravasation of tumor cells through heterologous gap junction formation with endothelial cells.

Among B16 mouse melanoma cells, F10 and BL6 cells are nearly identical except for their metastatic behavior (1). Both cell types colonize the lungs when injected into the tail vein (experimental metastasis assay). In contrast, when injected subcutaneously into the footpad (spontaneous metastasis assay), BL6 cells are much more metastatic than F10 cells (1). Since the spontaneous metastasis assay reflects the in vivo sequence of metastatic events, it appears that BL6 cells may possess the full complement of factors essential for metastasis, all or some of which may be missing from F10 cells. Alternatively, F10, but not BL6 cells, may express a factor that suppresses metastasis. Genes encoding such factors are likely to be differentially expressed between the two cell lines. We have reported previously an improved method for constructing subtracted cDNA libraries (2–4). Application of this method to F10 and BL6 cells yielded a set of genes that were transcriptionally upregulated in BL6 cells (5, 6). Here, we report that connexin 26 (Cx26) is one of these genes and may regulate the metastatic phenotype of melanoma cells.

Connexins are a family of at least 12 proteins that make up the intercellular channels of gap junctions (7). Gap junction hemichannels, called connexons, are composed of homomeric or heteromeric hexamers of connexins and provide conduits for the direct passage of ions and other low-molecular-weight molecules, including second messengers, between cells (8). This type of intercellular communication is the essence of direct contact–mediated cell-growth control and is important for many physiologic and developmental processes (9–11). Several reports have shown that gap junctional intercellular communication (GJIC) is disturbed during carcinogenesis (12–16). On the other hand, the role of connexins in the acquisition of more malignant phenotypes remains controversial (17–20).

Cx26 expression has been examined intensively in hepatocytes and squamous epithelial cells under physiologic and pathologic conditions (15, 21–24). The expression level of Cx26 is believed to decrease as hepatocytes and keratinocytes acquire the potential to proliferate autonomously (21, 23). We reported that transfection with Cx26, but not with Cx40 and Cx43, produced an inhibitory effect on the in vitro and in vivo growth of HeLa squamous cell carcinoma cells (15). On the other hand, the role of Cx26 during carcinogenesis of melanocytes and the malignant progression of melanoma cells is poorly understood. In the present study, we found that BL6 cells expressed Cx26 as abundantly as liver tissues. Subsequently, BL6 cells, but not F10 cells, were found to constitute a GJIC-competent cell population. Interestingly, BL6 cells could readily form a heterologous gap junction with endothelial cells, but could not form them efficiently with each other. The spontaneous metastasis assay indicated that the formation of such heterologous gap junctions by Cx26 might be responsible for the successful metastasis of F10 and BL6 cells.

Cx26-dependent dye coupling between melanoma and endothelial cells. While examining the difference in gene expression levels between F10 and BL6 cells by the cDNA subtraction method, we found that Cx26 gene expression was upregulated by fivefold in BL6 cells (Figure 1a). Consistently, BL6 cells expressed a significantly higher level of the Cx26 protein compared with F10 cells (Figure 1b). In B16, which is the parental strain of both F10 and BL6 cells, the expression level of the Cx26 gene and protein was similar to that of F10 cells (data not shown). The mRNA levels for other connexins were below the limit of detection in both F10 and BL6 cells (Figure 1a). Five clones carrying the full-length Cx26 cDNA were isolated from a BL6 cDNA library and sequenced. No mutations were detected in their coding regions. We performed the dye-transfer assay to examine whether GJIC ability increased in BL6 cells. Under in vitro culture conditions, neither F10 nor BL6 cells showed any GJIC ability by themselves (data not shown). We also examined dye transfer from melanoma to NIH3T3 fibroblastic monolayer cells and from melanoma to SVEC4-10 or HUV-EC-C endothelial monolayer cells under in vitro culture conditions. Here again, little or no dye coupling was detected (data not shown).

Figure 1

Expression of various connexins in F10 and BL6 cells and in the IVC segment. (a) Northern blot analysis to detect the transcripts of Cx26, Cx32, Cx43, Cx37, and Cx40. As a positive control, mouse liver, heart and lung tissues, and SVEC4-10 cells were used. (b) Expression of Cx26 protein in F10 and BL6 cells. Cell lysates were extracted by alkaline treatment. Anti-Cx26 antibody detected monomeric Cx26 protein at approximately 24 kDa (indicated by an arrow in the right panel). Equal protein loading was confirmed by silver staining of the gel (left panel).

Hence, we devised a coculture in which we placed an opened vein segment, with its luminal surface uppermost, at the bottom of a culture dish and seeded melanoma cells onto it. We investigated whether opened IVC segments preserved the integrity of the endothelial cell monolayer during cultivation. At the start of cultivation, only the luminal surfaces of the IVC segments located close to the cut edges were stained with trypan blue, but the central regions (above 80% of the total surface) were not (data not shown). This was the case for IVC segments after 2–6 hours of cultivation (data not shown). To further identify the endothelial cells, transverse sections of the IVC segments were reacted with antibodies against PECAM-1 and integrin αv, two proteins specifically expressed on endothelial cell membranes. Both antibodies stained the luminal surface of the IVC segment (Figure 2, a and b). In addition, the reactivity of the anti-integrin αv antibody drew a line that paralleled the luminal surface, corresponding to the endothelial basal membrane (Figure 2b). These results indicated that IVC segments retained a continuous endothelium in their uppermost layers despite the occasional absence of nuclei. The VE-cadherin antibody preferentially stained the endothelial luminal membranes; some staining appeared to locate at the site of endothelial cell-cell attachment (Figure 2c). Paxillin staining was detected as punctate inside the endothelial cells and mainly overlapped with integrin αv staining (Figure 2d). Northern blotting using the IVC segment revealed that the mRNA levels for connexins other than Cx43 were below the limit of detection (Figure 1a). The Cx43 protein was located widely in the endothelial cytoplasm, but some staining was concentrated at the site of endothelial cell-cell attachment (Figure 2e). The immunoreactivity of endothelial cells did not change after 6 hours of cultivation (data not shown). No specific immunostains were detected in any negative control experiments (data not shown). These results suggested that endothelial cells preserved their integrity with respect to both cell-cell and cell-extracellular matrix interactions throughout the culture periods.

Figure 2

Representative transverse sections of the IVC segment after culturing for 4 hours. Sections in the left panels were reacted with antibodies against PECAM-1 (a), integrin αv (b), VE-cadherin (c), paxillin (d), and Cx43 (e), and stained with FITC. Adjacent sections were stained with H & E (right side). Endothelial cells locate in the intima, and smooth muscle cells locate in the media of IVC. Pericytes may be present in the boundary between the intima and media. H & E stains nuclei of these cells dark blue. Arrows in b and d indicate the staining on the endothelial basal membranes. Arrows in c and e indicate the staining (left side) at the site of endothelial cell-cell attachment. Bar: 20 μm.

When F10 or BL6 cells were seeded onto the opened IVC segments, most melanoma cells settled down on the IVC surface within 2 hours. Consistent with the result of Western blot (Figure 1b), BL6 cells, but not F10 cells, expressed Cx26 during the coculture period (Figure 3, a and b). Some immunostaining was concentrated on the BL6 cell membrane facing the vein (Figure 3b). Next, we seeded BCECF-labeled melanoma cells onto the IVC segment and observed the dye-transfer profile directly above the coculture with a microscope equipped for epifluorescence. At 2 hours of coculture, all the F10 and BL6 cells displayed strong fluorescence in their cytoplasm, demonstrating the absence of dye transfer (Figure 3, c and d). When we scanned the coculture at 4 hours, we occasionally found a weakly fluorescent area around the BL6 cells, but not around the F10 cells (Figure 3e). At higher magnification, dye transfer from BL6 cells into endothelial cells was clearly demonstrated (Figure 3f). The dye was also transferred between neighboring endothelial cells. At 6 hours, most BL6 cells lost their fluorescence, whereas most F10 cells remained as fluorescent as those at 4 hours (data not shown).

Figure 3

Dye-transfer assay using the coculture composed of melanoma cells and IVC segments. (a and b) Cx26 expression in melanoma cells after culturing on IVC for 4 hours. Transverse sections of the cocultured tissue containing F10 (A in panel a) or BL6 (B in panel b) cells were stained with anti-Cx26 antibody. Arrows indicate the localization of Cx26 protein on the cell membrane. Also, note that endothelial cells of IVC contain much less Cx26 than Cx43. Bar: 20 μm. (c–f) BCECF-labeled F10 (c and e) or BL6 (d and f) cells were seeded onto an IVC segment and observed from directly above the coculture at 2 (c and d) and 4 (e and f) hours using a microscope equipped for epifluorescence. The relative intensity of fluorescence is displayed by the 256-color spectrum; red indicates a greater intensity than blue. Arrowheads indicate BCECF-labeled F10 and BL6 cells. Bar: 20 μm. (g) The number of cells showing dye coupling with endothelial cells was counted in a sample of 500 BCECF-labeled cells observed after 4 hours of coculture. Data are expressed as the mean of three independent experiments. Bars = SE; SE was sometimes too small to be represented. ^AP < 0.01 by t test when compared with the value obtained from F10 cells. ^BP < 0.01 by t test when compared with the value obtained from BL6 cells.

To examine this phenomenon more systematically, we prepared five dishes containing cocultures of F10 or BL6 cells with IVC segments. Each cocultured tissue was fixed with glutaraldehyde every 15 minutes from 3 hours 30 minutes to 4 hours 30 minutes. In each coculture, we examined 500 BCECF-labeled F10 or BL6 cells seeded onto the IVC segment. At 4 hours of coculture, on average, 22 BL6 cells showed dye coupling with endothelial cells (Figure 3g). When we observed the cocultured tissues fixed at other intervals, the number of BL6 cells showing dye coupling with endothelial cells was similar to that at 4 hours (data not shown). In contrast, little or no dye coupling was observed in any F10 cell-containing cocultures.

Various F10 and BL6 clones were obtained by transfection with wild-type and mutant forms of Cx26 and Cx32 (Figure 4b, blot). The C60F, P87L, and R143W mutations of Cx26 locate in the first extracellular and second and third transmembrane domains, respectively, and exert different dominant-negative effects on Cx26 (25). The former abrogates Cx26-mediated GJIC and the latter two abrogate Cx26-mediated cell growth control in HeLa cells (25). Transfectant clones were examined for GJIC ability with endothelial cells according to the protocol above. Figure 3g showed the results of the 4-hour coculture; the results at the other four intervals were essentially similar. F10-Cx26^WT-1, -2, and -3 cells showed dye coupling as frequently as BL6 cells. Transfection with the C60F or R143W-mutant form did not elevate the dye-coupling ability of F10 cells, whereas transfection with the P87L-mutant form produced a few dye coupling–competent F10 cells. Thus, the former two mutant forms were introduced into BL6 cells to examine whether these mutant forms abrogate the dye-coupling competency of BL6 cells. Among cells of two BL6-Cx26^C60F and three BL6-Cx26^R143W clones, only a few showed dye coupling, indicating a dominant-negative effect of the C60F and R143W mutations in this assay. F10-Cx32^WT-1 and -2 cells were deficient in dye coupling.

Figure 4

Spontaneous metastasis assay of Cx26 and Cx32 transfectants. (a) Primary tumor growth at the inoculation site (right footpad). Growth curves for the five representative clones are expressed as the mean and SE of three independent experiments. The values of the other clones used were within the range of these five clones at any time point. There was no significant difference between the clones. (b) The number of colonies in the lungs produced by various F10 and BL6 clones. F10-pcDNA3 and BL6-pcDNA3 clones are vector controls. Data are expressed as the mean and SE of three independent experiments. ^AP < 0.05 by t test when compared with the value obtained from F10 cells. ^BP < 0.05 by t test when compared with the value obtained from BL6 cells. The Northern blot shows the detection of endogenous and exogenous Cx26 and Cx32 mRNA in each clone. (c) Representative results of the lung metastatic colonies produced by F10, BL6, F10-Cx26^WT-1, and BL6-Cx26^C60F-1 cells. Note that BL6 and BL6-Cx26^C60F-1 cells produced solid black colonies whereas F10-Cx26^WT-1 cells produced amelanotic colonies as well as melanotic ones.

Metastatic phenotype of Cx transfectants. We examined the metastatic proficiency of dye coupling–competent F10 clones and dye coupling–deficient BL6 cells. The original melanoma cells and their transfectants were injected subcutaneously into mouse footpads. Tumor growth at the injection sites was the same among all the clones examined (Figure 4a). F10 clones transfected with wild-type Cx26 were more metastatic than untransfected F10 cells, and transfection with mutant forms of Cx26 did not alter the metastatic phenotype of F10 cells (Figure 4b). Some lung colonies produced by F10-Cx26^WT clones looked amelanotic, which contrasted with the solid black appearance of BL6-derived colonies (Figure 4c). C60F and R143W mutations produced an inhibitory effect on the spontaneous metastasis of BL6 cells. Of the five dye coupling–deficient BL6 clones, BL6-Cx26^C60F-1, BL6-Cx26^R143W-1, and -3 cells displayed threefold less metastatic colonies compared with the original BL6 cells (Figure 4, b and c). On the other hand, the reduction in the number of metastatic colonies by other BL6 clones was small. There was the possibility that the in vivo expression levels of Cx26 differed from that of the in vitro levels. We examined the Cx26 gene expression in the tumors of various clones growing in footpads and lungs and found that the expression level for the endogenous and exogenous Cx26 gene in each clone was essentially the same as that of cultured cells (data not shown).

Cx26 expression in human melanoma. The clinical relevance of Cx26 to malignant phenotypes of human melanocytic lesions was assessed by immunohistochemistry. We examined five cases with intradermal nevus, five cases with in situ melanoma (Clark’s level I), and seven cases with more advanced melanoma (Clark’s level III or IV). Nevus cells and in situ melanoma cells were Cx26 negative in all of the cases examined (data not shown and Figure 5, a and b). In the specimen of nevus, we observed the epidermis apart from the nevus lesion and found that melanocytes were also Cx26 negative under physiological conditions (data not shown). In the cases of advanced melanoma, immunoreactivity varied at sites containing melanoma cells. Melanoma cells resident within the epidermal basal layer were Cx26 negative or faintly positive in all seven cases examined (Figure 5, c and d). In contrast, melanoma cells detached from the basal layer or in the process of invading the dermis often showed Cx26-positive staining (Figure 5, c and d). This increase in the immunoreactivity of melanoma cells was detected in six of the seven cases.

Figure 5

Histologic examination of human melanoma tissues by staining with anti-Cx26 antibody (left side) and H & E (right side). Four representative cases are shown. (a andb) Two cases with in situ melanoma (Clark’s level = I). (c and d) Two cases with advanced melanoma (Clark’s level = III). Keratinocytes served as an internal control for immunohistochemical staining (21). Arrows indicate melanoma cells or lesions still resident within the epidermis, most of which were Cx26 negative. Note that the reactivity for Cx26 antibody increased in melanoma cells invading into the dermis, as seen in the left half of c and d. Bar: 75 μm.

Although Cx expression in melanocytes or melanoma cells has not been studied intensively, we found that BL6 cells expressed the Cx26 gene as abundantly as liver tissues. Hepatocytes are one of the cell types that express Cx26 most abundantly and exhibit an efficient dye coupling with one another (32, 33). Since there was no mutation in the coding sequence of the Cx26 mRNA expressed in BL6 cells, BL6 cells were expected to be as GJIC proficient as hepatocytes. Unexpectedly, we could detect little dye coupling between BL6 cells in culture. Also, little or no dye transfer was detected from BL6 cells into endothelial cells cultured on dishes. Instead, we detected dye transfer from BL6 cells into endothelial cells making up the IVC. In addition to BL6 cells, F10 cells acquired the similar dye-transfer ability by transfection with Cx26. Transfection with dominant-negative forms of Cx26 abrogated the dye-coupling proficiency of BL6 cells. These results indicated that dye-coupling between melanoma and endothelial cells was a Cx26-specific event. However, the nature of the connexins on endothelial cells that participate in the formation of gap junctions with Cx26-expressing melanoma cells remains unclear. There was little, if any, Cx26 expression in the IVC segments at either the mRNA or protein level (Figure 1a and Figure 3, a and b). It has been shown that a heterotypic gap junction between Cx32 and Cx26 transferred dye as efficiently as a Cx26-homotypic gap junction (34). However, it is not likely that Cx32 on endothelial cells is a counterpart of Cx26 on melanoma cells, since Cx32-transfected F10 clones were deficient in dye coupling with endothelial cells. Endothelial cells have been known to express several Cx subtypes, including Cx37, Cx40, and Cx43 (35). We detected the Cx43 gene expression in the IVC segments (Figure 1a) and determined the Cx43 protein localization in endothelial cells of IVC (Figure 2e). However, the Cx26-Cx43 heterologous gap junction is insufficient for communication between Cx-transfected HeLa cells in culture (34). Efficient formation of GJIC requires the integrity of both cell-cell and cell–extracellular matrix interactions (36–38). Since endothelial cells of the IVC segment appeared to preserve this type of integrity (Figure 2) they could communicate with melanoma cells through the Cx26-Cx43 heterologous gap junction.

At most, 5% of the BL6 cells seeded onto IVC segments transferred dye into endothelial cells at a given time point. This raises the possibility that only a portion of the BL6 cell population was able to transfer dye. The following results may throw some light on this issue: (a) at 6 hours, most BL6 cells had lost their fluorescence, whereas most F10 cells remained as fluorescent as those at 4 hours; (b) the expression level of the Cx26 gene in BL6 cells did not change even after their metastasis into the lung; and (c) none of the transfectant clones of BL6 displayed altered expression levels of the endogenous Cx26 gene (Figure 4b). These results do not exclude, but rather favor, the interpretation that BL6 cells were a dye coupling–competent cell population. On the other hand, F10 cells rarely showed dye coupling with endothelial cells at any time point, indicating that F10 cells are a dye coupling–deficient cell population.

We examined whether such a difference in GJIC ability might be involved in metastasis of F10 and BL6 cells. All the three dye coupling–proficient F10 clones, obtained by transfection with wild-type Cx26, were as spontaneously metastatic as BL6 cells. Three out of five dye coupling–deficient BL6 clones, obtained by transfection with dominant-negative forms of Cx26, significantly reduced their spontaneously metastatic potential. On the other hand, the reduction in the number of metastatic colonies was small in the two BL6 clones of the rest. This may have been due to the lower expression of C60F and R143W dominant-negative forms in these clones in vitro (Figure 4b, blot). Although there were no direct data to indicate how Cx26 might exert its effects in vivo, the results above suggested an involvement of heterologous gap junction between melanoma and endothelial cells in spontaneous metastasis. F10 cells are as proficient as BL6 for the colonization of the lung after intravenous injection, but deficient after subcutaneous injection (1). The metastatic behavior of BL6 cells appears to differ from that of F10 cells before or during the process of tumor cell intravasation at the primary site. Cx26 may function during the process of intravasation around the primary sites. This is reminiscent of a report that demonstrated GJIC formation between leukocytes and endothelial cells at the site of leukocyte permeabilization of blood vessels (39). Melanoma cells are likely to use the same GJIC-dependent mechanism to enter the blood vessel (40, 41). This type of heterologous GJIC might trigger the endothelial cell dissociation at the site of a tumor cell–endothelial cell interaction (42). However, our data could not rule out Cx26 involvement in other stages of metastasis.

Cx26 expression was low not only in melanocytes and nevus cells, but also in melanoma cells that were still resident in the epidermal basal layer. The results were consistent with the fact that Cx26 has been characterized as a carcinogenesis suppressor (11, 15). In contrast, melanoma cells markedly increased their expression level of Cx26 after detachment from the epidermis and in the process of invading the dermis (Figure 5). Kamibayashi et al. (21) reported the decline of Cx26 expression in the course of development of carcinogenesis from papilloma to squamous cell carcinoma, another type of skin tumor originated from keratinocytes. Interestingly, they also demonstrated that Cx26 expression apparently increased in the cancer cells metastasizing into lymph nodes. Their results and ours consistently suggest a possibility that once tumor cells have acquired autonomous growth potential and detached from the primary sites, higher expression of Cx26 may promote invasion and metastasis.

Several recent reports support this speculation. Vaccines prepared from Cx37-derived peptides have been reported to protect mice from spontaneous metastasis of Lewis lung carcinoma (43). Transfection with Cx43 increased the invasive potential of HeLa cells on heart cells after formation of heterotypic dye coupling between both cells (44). Here, we found a correlation in melanoma cells between their metastatic phenotypes and GJIC proficiency. Malignant glioma ranks with malignant melanoma as a tumor with a very poor prognosis (45, 46). Recently, functional gap junctions were found to form between glioma cells and surrounding astrocytes, resulting in the phenotypic transformation of the astrocytes (47). This kind of Cx function might be called “acclimation.” Both glioma and melanoma cells are able to communicate directly with distinct types of normal host cells. Glioma cells, characterized by their diffuse infiltration into surrounding brain tissue (45), are coupled with astrocytes. Melanoma cells, characterized by a high incidence of metastasis (46), are coupled with endothelial cells. Such couplings might contribute to the susceptibility of the host to tumor invasion.

We thank Kenji Morihana and Akinori Fukuyama for technical assistance with histologic experiments. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan and grants from the Osaka Cancer Society, The Naito Foundation, and Seijinbyo-igaku Foundation.

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