Connexin 43 mediated gap junctional communication enhances breast tumor cell diapedesis in culture

Human microvascular endothelial cells (HMVECs) derived from the lung were purchased from Cambrex Biosciences Inc. (Walkersville, MD, USA) and cultured according to supplier's instructions in endothelial growth medium (EGM; Cambrex Biosciences Inc.), supplemented with 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulphate (Invitrogen, Burlington, ON, Canada). Wild-type transformed mammary epithelial cells (HBL100) and HBL100 cells expressing Cx43 or Cx43 with green fluorescent protein (GFP) tagged to the amino terminal (GFP-Cx43) [33] were routinely cultured in D-MEM supplemented with 100 units/ml penicillin G sodium, 100 μg/ml streptomycin, 2 mM L-glutamine (Invitrogen) and 10% FBS (Sigma-Aldrich, Oakville, ON, Canada). All cells were maintained in a humidified chamber at 37°C containing 5% CO₂.

HBL100 cells expressing Cx43 (HBL100Cx43) or empty vector (HBL100v) were engineered by retroviral infection as described by Qin et al. [34]. Briefly the cDNA of Cx43 inserted into the AP-2 retroviral vector was transfected into the 293GPG packaging cell line and 48 h later retroviral supernatant was collected and filtered through a 0.45 μm filter and used to infect HBL100 cells. Twenty-four hours after infection, medium was replaced with D-MEM and cells were subcultured as normal. More than 90% of tumor cells expressed Cx43 after three rounds of viral infection as determined by immunofluorescence [34].

HBL100 cells expressing non-functional GFP-Cx43 (HBL100GFP-Cx43) [33] were generated by transfecting 2.5 × 10⁵ HBL100 cells with 2 μg of GFP-Cx43 using Metafectene (Biontex Laboratories GmbH, Munich, Germany). After 24 h, approximately 40% of cells expressed GFP-Cx43 and these cells were used for diapedesis assays.

The diapedesis assay was carried out as previously described [9]. Briefly, 7.5 × 10⁵ HMVECs were seeded onto 12 mm glass coverslips coated with matrigel (Becton-Dickenson, Bedford, MA, USA). HMVEC cells were allowed to settle for 2–3 h and form monolayers in a 37°C humidified chamber containing 5% CO₂. Coverslips were then transferred to a 24-well plate and cultured for 48 h in EGM. The number of cells seeded produces an endothelial monolayer without requiring cell proliferation. Monolayers have intact adherens junctions as previously evaluated by VE-cadherin staining [9, 35]. HBL100 tumor cells were labeled with 10 μg/ml 1,1'-dioctadecyl-3, 3,3', 3'-tetramethylindocarbocyanine percholate (DiI; Molecular Probes, Eugene, OR, USA), and harvested non-enzymatically using 2 mM EDTA (EMD Chemicals Inc., Gibbstown, NJ, USA). Approximately 7.5 × 10⁴ cells were added to endothelial monolayers at a ratio of around 1:10 (tumor cell:endothelial cell). Cells were then co-cultured for different times (1, 5, or 7 h) before live observation or fixation and immunostaining. Diapedesis was quantified blinded to the investigators using a Leica IRE2-DM epifluorescence microscope (Leica Microsystems Inc., Toronto, ON, Canada) as previously described [11]. Briefly, co-cultures were fixed and stained for F-actin. Diapedesis was quantified by counting the number of tumor cells in contact with the endothelium classified into three stages according to their position relative to the endothelium: round on top (tumor cells with a spherical shape located on the apical surface of the endothelium); migrating (tumor cells have penetrated through the endothelial monolayer at endothelial cell junctions where part of the cell body is located above the endothelium and part of it has spread on the matrigel, beneath the endothelial cell f-actin stress fibers); and underneath (all of the tumor cell body is below the plane of the endothelial cell stress fibers). Migrating and underneath cells were scored together as transmigrating. For each coverslip, approximately 100 cells adherent to the endothelium were scored and counted and all experiments were repeated three times with triplicate coverslips.

GJIC was evaluated using the preloading dye coupling assay as described previously [36] with the following modifications. Tumor cells were labeled for 15 minutes at 37°C with 2 ml of Opti-MEM (Invitrogen) containing 10 μg/ml calcein-AM and 10 μg/ml DiI. Calcein-AM is a fluorescent substrate that is cleaved in viable cells into a membrane impermeable form able to pass through functional gap junctions but not through other plasma membrane channels. Labeled tumor cells were washed twice with Hank's balanced salt solution (HBSS), harvested non-enzymatically using 2 mM EDTA, and added to HMVEC monolayers on matrigel. Dye transfer from tumor cells to endothelial cells was observed live by epifluorescence microscopy after 30 minutes of co-culture and the number of adherent tumor cells that transferred dye to adjacent endothelial cells was scored and expressed as percent of total number of tumor cells counted. For each coverslip, approximately 70 cells adherent to the endothelium were scored. Unless specified, all experiments were repeated three times with triplicate coverslips.

To determine whether carbenoxolone (CBX) blocks gap junctional coupling, HMVEC monolayers were pretreated for 7 h with 150 μM CBX or the inactive analog glycyrrhizic acid (GZA). HMVEC were then loaded for 15 minutes with 10 μg/ml calcein-AM (Molecular Probes) in an Opti-MEM solution containing CBX or GZA. Cells were rinsed twice with Opti-MEM containing CBX or GZA and immersed in fresh media containing CBX or GZA. Because the fluorescent dye can only pass through gap junctions, recovery of fluorescence in the bleached cell will only occur if dye passes through gap junctions with adjoining unbleached cells. For each experimental condition, three individual cells were bleached for approximately 30 s using the Zeiss laser scanning confocal microscope (LSM) 410 and an argon laser at full power. To observe recovery of fluorescence, images were obtained every five minutes following photobleaching using the argon laser at 33% power until maximum recovery was reached (15 minutes). In control experiments, monolayers were left untreated or were treated with the inactive analog of CBX, GZA. Mean fluorescence intensity of bleached/unbleached cells in the same area was measured over time using scion imaging software and expressed as mean relative recovery (arbitrary units). Data represent three independent experiments.

Cells were fixed at room temperature for 20 minutes with 2% or 4% (w/v) paraformaldehyde (EMD Chemicals Inc.) and washed in cation free phosphate buffered saline (PBS). Cells were permeabilized for five minutes at 4°C using a buffer containing 15 mM Tris-HCL, 120 mM sodium chloride (NaCl), 25 mM potassium chloride (KCl), 2 mM EDTA, 2 mM EGTA, 0.5% (v/v) Triton X-100, pH 7.5. To label F-actin, co-cultures were incubated for 30 minutes at room temperature with Alexa Fluor^® 488 or Texas Red conjugated-phalloidin (Molecular Probes) diluted 1:50 in PBS with 1% (w/v) bovine serum albumin (BSA) followed by three washes in PBS. To examine the localization of F-actin and Cx43, cells were labeled with Texas red conjugated-phalloidin (1:50 dilution) and with monoclonal anti-Cx43 (clone P4G9 from the Fred Hutchinson Cancer Research Center Antibody Development Group, Seattle, WA, USA) diluted 1:10 in 1% (w/v) BSA/PBS. Cells were incubated with Alexa Fluor^® 488-conjugated goat-anti-mouse secondary antibody (Molecular Probes). Nuclei were stained using 10 μg/ml Hoechst 33342 (Sigma-Aldrich) diluted in PBS. Coverslips were mounted on glass slides using vectashield mounting media (Vector Laboratories, Burlington, ON, Canada) and sealed with nail enamel. The inclusion of spacers made from plastic coverslips cut into strips prevented contact of co-cultures with the slides as described by Voura et al. [37].

Co-cultures were imaged using a Zeiss LSM 410 laser scanning confocal microscope system (Carl Zeiss Canada Ltd, Toronto, ON, Canada). Serial 0.7 μm optical sections of selected areas were recorded and evaluated with the LSM 410 software (Carl Zeiss Canada Ltd). Representative serial optical sections are presented in an apical to basal direction.

Adhesion to the endothelium was evaluated as previously described for leukocyte adhesion to the endothelium [38] with the following modifications. HMVEC monolayers were established in 1:20 matrigel coated wells (2.1 × 10⁴ cells per well) and cultured for 48 h. HBL100, HBL100v, or HBL100Cx43 cells (2.1 × 10³), pre-labeled with DiI, were added to HMVEC monolayers and co-cultured for 1 h. Co-cultures were fixed with 2% (w/v) paraformaldehyde and washed twice with PBS to remove non-adherent cells. The PBS was replaced with water and fluorescence intensity of DiI was measured for each well at 549 nm excitation and 565 nm emission wavelengths using a SAFIRE fluorescent microplate reader (Tecan US Inc., Durham, NC, USA). The number of adherent tumor cells was calculated and normalized based on a standard curve generated with known numbers of DiI-labeled tumor cells present in duplicate wells in the same microplate. Experiments were repeated three times with eight sample replicates per cell type.

HBL100, HBL100v, HBL100Cx43, HBL100GFP-Cx43, and HMVEC cells grown in culture dishes were lysed using the following buffer for protein collection: 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% (w/v) SDS, and 0.5% (v/v) Triton X-100. Protein concentration was determined with bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical Co., Rockford, IL, USA). Protein (50 μg) for each cell type was separated in 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked with 5% non-fat dry milk in tris buffered saline (TBS) containing 5% Tween 20. Membranes were then probed for Cx43 protein, using a monoclonal anti-Cx43 antibody against amino-terminal amino acid residues 1–20 (clone P1E11 from the Fred Hutchinson Cancer Research Center Antibody Development Group, Seattle, WA, USA) at a dilution of 1:500. Immunoblots were probed with appropriate secondary antibodies conjugated to horse radish peroxidase (Pierce Chemical Co.) at a dilution of 1:10,000. The protein was visualized using Supersignal West Pico Chemiluminescent substrate (Pierce Chemical Co.) by exposing the immunoblot to Amersham Hyperfilm (Amersham Biosciences, Piscataway, NJ, USA) for 30 s.

Comparisons between cell types and different time points or treatments were evaluated using a two-way analysis of variance followed by Bonferroni post-hoc test. A p-value of < 0.05 was considered to be significant. Comparisons between cell types at a single time point were evaluated using one-way analysis of variance followed by Tukey's multiple comparison post-hoc test. Data are presented as means +/- SEM.

Previous work suggested that GJIC between tumor cells and endothelial cells may affect diapedesis during metastasis [15‐17]. To analyze and evaluate the efficiency of breast tumor cell diapedesis we used an in vitro assay in which tumor cells were allowed to migrate through a monolayer of endothelial cells grown on matrigel as we have previously described [9, 39‐41]. This assay mimics aspects of the vessel wall and has been used by us [35, 42] and others [39‐41] to analyze leukocyte diapedesis. Diapedesis was assessed by evaluating the spatial location of tumor cells as they migrated through an endothelial monolayer. Tumor cells pre-labeled with DiI were seeded for 1, 5 or 7 h onto endothelial monolayers before fixation and stained for F-actin. Tumor cells were scored according to their morphology and location with respect to the endothelium. We defined three distinct stages of migration observable by confocal microscopy (Fig. 1): top; migrating; and underneath. Tumor cells located on top of the endothelium prior to diapedesis tended to have a round or oval shape with small filopodia extending from the periphery of the cell (Fig. 1b, arrows). This shape was maintained from the apical (Fig. 1b) to the basal surface (Fig. 1d) of the tumor cell, differing from melanoma cells, which tend to have a fibroblastic shape and spread on the apical endothelial surface [37]. At sites of tumor cell-endothelial cell interactions, prominent microfilament bundles within the underlying endothelial cell were seen, indicating no disruption of the endothelium (Fig. 1d). Tumor cells in the process of diapedesis (migrating) had a unique morphology with distinct shapes at various focal levels (Fig. 1f–h) not previously seen in melanoma cells [37] but very similar to transmigrating monocytes and prostate tumor cells [11, 35]. The apical aspect of migrating tumor cells located above the endothelium was round in shape and lacking filopodial extensions, but contained smooth cortical actin staining (Fig. 1f). At the level of the endothelium, tumor cells maintained close contact with the endothelial cells, which formed a circular opening surrounded by thick bundles of F-actin (Fig. 1g, arrow). The basal aspect of the tumor cells was located underneath the endothelium and spread along the matrigel by extending processes in various directions (Fig. 1h, arrows). Tumor cells located completely underneath the endothelium (Fig. 1j–l) had endothelial microfilament bundles extended over the top of the tumor cell as previously seen in melanoma cells [37]. No opening above the tumor cell remained, indicating complete closure of the transmigration passage (Fig. 1j). Tumor cells underneath the endothelium spreading on the surface of the matrigel had an irregular shape and stress fibers (Fig. 1l, arrows). The morphological features of HBL100v or HBL100Cx43 cells (not shown) during diapedesis were not noticeably different from those of HBL100 wild-type cells. We used the morphological criteria shown in Fig. 1 to quantify various stages and assess the efficiency of diapedesis.

To explore if Cx43 expression in HBL100 cells affects any aspects of diapedesis, cells were engineered to express functional Cx43, non-functional GFP-Cx43 or the empty retroviral vector as a control. Cx43 was absent in both wild-type and empty vector HBL100v control cell lines used (Fig. 2a,c,d) but expressed in HMVEC (Fig. 2a,b) and cells engineered to express Cx43 (Fig. 2a,e) or GFP-Cx43 (Fig. 2a,f). Importantly, both Cx43 and GFP-Cx43 were routinely observed to localize to the cell surface, consistent with the formation of gap junction plaques (Fig. 2b,e,f). Intracellular populations of Cx43 likely reflecting different stages of assembly or degradation in HMVEC, HBL100Cx43 and HBLGFP-Cx43 were also seen. These results suggest that exogenous or endogenously expressed Cx43 assembles into gap junctional plaques at sites of homocellular endothelial or tumor cell-cell contacts.

To determine whether HBL100 cells engineered to exogenously express Cx43 formed gap junctions and established GJIC with HMVEC cells, we co-cultured HBL100Cx43 with HMVEC monolayers, localized Cx43 in these cultures (Fig. 3a–c), and assayed for dye transfer between tumor and endothelial cells (Fig. 3d–j). Confocal microscopy revealed Cx43 at tumor cell-endothelial cell interfaces (Fig. 3b, arrows) where tumor cells were wedged between endothelial cells and where cortical F-actin was found (Fig. 3b,c). These results indicate that during diapedesis HBL100Cx43 cells form heterocellular gap junctions with adjacent endothelial cells.

Preloading dye transfer studies to assess whether Cx43 is being assembled into functional gap junctions at tumor/endothelial cell interfaces revealed that dye spread extensively from Cx43 expressing HBL100 cells to endothelial cells (Fig. 3i), whereas dye failed to spread from control HBL100v cells to endothelial cells (Fig. 3f). These results suggest that exogenous Cx43 in HBL100Cx43 can form functional gap junctions with HMVEC early on during diapedesis. Live quantification of coupling performed after 30 minutes of co-culture revealed that ten times more Cx43 expressing HBL100 cells coupled to the endothelium than those lacking Cx43 (Fig. 3j). The low occurrence of dye transfer between HBL100v control cells and HMVEC is likely due to nonspecific dye uptake from damaged tumor cells rather than functional gap junctional coupling due to connexin expression.

To compare the efficiency of diapedesis of Cx43 deficient tumor cells with those that exogenously express Cx43, we co-cultured HBL100, HBL100v or HBL100Cx43 cells with HMVEC for 1, 5 and 7 h. At all time points HBL100Cx43 cells showed an up to two-fold increase in diapedesis efficiency compared to cells lacking Cx43 (Fig. 4a). This increase in migration appeared to plateau at 5 h. Conversely, at all time points no statistical difference in migration efficiency was observed between wild-type (HBL100) and control (HBL100v) cells (Fig. 4a). The relative proportion of cells at different stages of migration was evaluated after 7 h of co-culture (Fig. 4b). About 50% of wild-type and vector control cells encountered were located on top of the endothelium, 40% were migrating across the endothelium, and about 10% were completely located underneath. In contrast, only 20% of HBL100Cx43 cells were located on top of the endothelium, while over 60% migrated across. No significant difference in the relative proportion of cells underneath the endothelium was observed between HBL100 cells with or without Cx43 expression (Fig. 4b). These results suggested that Cx43 facilitated tumor cell diapedesis but it was unclear if this was due to increased adhesiveness or whether GJIC was required.

To determine whether the increase in diapedesis efficiency was due to increased adhesive properties of Cx43 expressing tumor cells, we co-cultured HBL100, HBL100v and HBL100Cx43 cells on endothelial cells and compared the number of DiI labeled tumor cells adherent to HMVEC monolayers using fluorescence intensity measurements. No significant difference in the ability of tumor cells to adhere to the endothelium was observed between HBL100 breast tumor cells expressing Cx43 and tumor cells lacking Cx43 expression (Fig. 5), suggesting that the increased efficiency of diapedesis was not due to increased cellular adhesion.

To determine whether GJIC was required to increase HBL100 tumor cell diapedesis, we chemically blocked gap junctional coupling using CBX. HMVEC monolayers were preloaded with calcein dye and fluorescence recovery after photobleaching (FRAP) analysis was first used to determine whether CBX effectively blocked gap junctions in endothelial cells. Individual cells within the calcein-AM loaded monolayer were bleached and recovery of fluorescence was quantified. Our results demonstrated that, in the presence of 150 μM CBX, fluorescence recovery in the bleached cell was prevented (Fig. 6a, triangles), indicating lack of homocellular gap junctional coupling. In GZA treated monolayers (negative control), however, recovery of fluorescence occurred at levels similar to those in untreated cells (Fig. 6a, rectangles and circles, respectively), indicating functional homocellular gap junctional coupling between adjacent endothelial cells.

To determine whether CBX would impair the Cx43-induced increase in diapedesis of HBL100Cx43 cells, HMVEC monolayers were pretreated with 150 μM CBX, or 150 μM GZA prior to adding tumor cells (Fig. 6b). In the presence of GZA HBL100Cx43 cells were more efficient at diapedesis compared to HBL100v control cells. A significant decrease in diapedesis efficiency in the presence of the gap junctional blocker CBX, however, was observed for both HBL100v and HBL100Cx43 cells. This finding suggests that gap junctional coupling between endothelial cells partially regulates diapedesis efficiency of tumor cells regardless of their Cx43 expression.

To determine if GJIC was necessary for Cx43-linked increase in diapedesis and, more specifically, if heterocellular GJIC was necessary between tumor and endothelial cells, we compared diapedesis of HBL100 cells that expressed either the functional or non-functional Cx43 (GFP-Cx43). Previous studies showed that GFP fused to the amino terminus of Cx43 (GFP-Cx43) results in a chimeric connexin that can traffic and form gap junction-like structures at cell-cell contacts, but these structures are not functional [33] and also functional hemichannels do not form [43]. HBL100, HBL100v, HBL100Cx43, or HBL100GFP-Cx43 cells were co-cultured with HMVEC for 7 h and scored according to the described criteria for stages of migration (Fig. 1). After 7 h of co-culture the percentage of migrating HBL100GFP-Cx43 cells was similar to that of migrating HBL100 and HBL100v cells, whereas diapedesis efficiency of HBL100Cx43 cells was more than 50% higher (Fig. 7). This result suggests that the presence of Cx43 protein in the tumor cell alone is not responsible for the observed increase in diapedesis, but that heterocellular GJIC between tumor cells and endothelial cells is required to augment the efficiency of diapedesis of HBL100 cells.