Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium
© Elzarrad et al; licensee BioMed Central Ltd. 2008
Received: 13 June 2008
Accepted: 22 July 2008
Published: 22 July 2008
The modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium.
Tumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 or dominant-negatively mutated connexin-43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function.
Adhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin-43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci.
Connexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis.
Intercellular communication via gap junctions between neighboring cells contributes to the organization of cells into functional tissues. Malignant tumors arising from planar epithelial tissue lose the normal growth limit caused by cell-cell contacts and form solid tumors in three dimensions. This observation was the origin of the notion that the loss of gap junctional intercellular communication (GJIC) plays an important role in tumorigenesis [1, 2]. Moreover, since invasive tumors are characterized by cell detachment from the primary mass, a disruption of intercellular junctions also became a de facto requirement for metastasis [3, 4].
A tumor cell undergoes a variety of stages in its life cycle: its origin and growth in the primary site, cellular detachment and intravasation, dissemination, settling in a new site by endothelial attachment, and its growth as a metastatic tumor including the development of its blood supply. While some of these stages, such as uncontrolled cell division and cellular detachment, are associated with loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, demand increased cell-cell contact. Therefore, it is clear that a simplistic notion of the loss of cellular communication cannot fully describe the status of the cellular interaction between tumor cells or between endothelial and tumor cells during the whole metastatic process. An adhesive interaction of tumor cells to endothelial cells during 'homing' is obvious. In addition, a role for connexins in the facilitation of tumor growth, metastatic potential, and tumor vascularization has been documented [5–12].
Does connexin-43 (Cx43) mediate GJIC between 4T1-GFP cells and pulmonary microvascular endothelial cells (PMVECs)?
Does Cx43 play a role in the attachment of breast cancer cells to endothelial cells in the pulmonary circulation?
Is Cx43 upregulation associated with the sites of micrometastatic foci and metastatic tumor vasculature?
To understand the role of Cx43 in the metastatic process, we used cancer cell lines that overexpress wild-type Cx43, express a dominant-negative mutant of Cx43, and a control mutant that does not affect endogenous Cx43 functions. Using these Cx43 mutant cell lines, we show that in a syngeneic mouse breast cancer experimental metastasis model, Cx43 enhances breast cancer cell attachment to the pulmonary endothelium. The results also indicate that Cx43 can serve as a marker of micrometastases and tumor vasculogenesis because of its significant upregulation in areas of tumor cell-endothelial cell contact areas in vitro and in vivo.
Cell culture and stable expression of Cx43 mutants
Pulmonary microvascular endothelial cells were obtained from the Endothelial Cell Culture Core at the Center for Lung Biology . These cells are routinely obtained by primary isolation from Sprague-Dawley rats and are used within passage 12. These cells are maintained and propagated in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum.
The 4T1 cells (a mouse breast adenocarcinoma cell line) were obtained from ATCC and were transfected with enhanced green fluorescent protein (eGFP) vector (Clontech, Mountain View, CA). Cells with stable GFP expression were separated by fluorescence activated cell sorting and propagated and maintained in DMEM with 10% fetal bovine serum. We have used the 4T1-GFP cells previously in both experimental and spontaneous metastasis assays [14–16]. Cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin and were maintained at 37°C in a moist environment of 95% air and 5% CO2 and sub-cultured as required.
The Cx43 missense mutation G138R was made by site-directed mutagenesis by changing the glycine to arginine on position 138 in the amino acid sequence. The G138R mutant expresses a mutant Cx43 that exhibits a dominant-negative effect when expressed in cells with endogenous Cx43 . This mutation is known to be present in patients suffering from oculodentodigital dysplasia [18, 19]. The expressed Cx43-G138R protein translocates to the membrane, and assembles as connexons, but does not exhibit functional GJIC. Cells expressing the G138R mutation exhibited a strong suppression of dye-transfer even when they were co-expressed with wild-type Cx43 . The G138R construct was ligated to a eukaryotic expression vector containing the zeocin resistance gene driven by the SV40 promoter. On the other hand, the C61S mutant Cx43 protein lacks gap junction channel forming abilities because of its cytoplasmic localization, and apparently it does not interfere with functional gap junction formation by endogenous normal Cx43 . The Cx43 point mutation C61S was generated by site-directed mutagenesis, replacing the cysteine residue in position 61 with a serine residue. The C61S construct was primarily used as a control. Both the C61S construct and the cDNA for wild-type Cx43 (Cx43 overexpressors, or Cx43OE) were ligated into the expression vector pBEHpac18. 4T1-GFP tumor cells were also transfected with the vector expressing two copies of wild-type Cx43 (Cx43OE) to enhance the formation of functional gap junctions in tumor cells (positive control).
Plasmid DNA was extracted from filter paper spots into 500 μl of Tris-EDTA buffer (10 mM Tris-Cl, pH 7.5 and 1 mM EDTA), electroporated into competent bacteria, and allowed to grow overnight with the appropriate antibiotic for selection. The plasmid DNA was isolated using Miniprep (Qiagen) and samples were run on a gel to confirm the size of the plasmid. In addition, samples of the plasmid DNA were sent to the GeneLab at Louisiana State University (Baton Rouge, LA) for amplification and sequencing. The presence of each mutation was confirmed by comparison with wild type using BLAST software.
4T1-GFP cells were grown to 75–95% confluency in 60 mm culture dishes and transfected at 37°C in Opti-MEM medium (Invitrogen, Carlsbad, CA) containing 75 μl of Lipofectamine 2000 and 30 μg of plasmid DNA. After 24 hours, the DNA/Lipofectamine suspension was removed and replaced with culture medium containing the appropriate antibiotics. Cells were selected with 2 μg/ml puromycin (InvivoGen, San Diego, CA) for the C61S and Cx43OE constructs or with 500 μg/ml of zeocin (Invitrogen) for the G138R construct. The selection process continued for 2 weeks on the puromycin resistance constructs and for 4 weeks on the zeocin resistant construct. Cells were maintained afterwards in normal medium containing the appropriate amounts of antibiotics.
Evaluation of gap junctions by dye-transfer assays in vitro and in situ
The presence of functional gap junctions was evaluated using a fluorescent dye-transfer assay. Sub-confluent monolayers of PMVECs in glass-bottomed 35 mm dishes were used. Endothelial cells were washed with Kreb's Ringer bicarbonate solution (KRB), with 5% clinical grade dextran and 10 mM glucose at pH 7.4, and incubated with 5 μM Calcein Orange Red AM (gap junction-permeable fluorescent dye, molecular weight 790 Da) in KRB for 30 minutes. The acetoxymethyl ester (AM) group of the dye is hydrolyzed by esterases in a viable cell, rendering the dye polarized and trapped within the cell, but allowing its transfer to adjacent cells via functional gap junctions. After dye loading, the endothelial cells were washed five times to remove the free dye from the medium. Experiments were performed in KRB in the absence of serum. Then 4T1-GFP cells (transferred-dye acceptors) were placed on the labeled endothelial cells (dye donors) at a ratio of 1:500 (acceptor:donor). After incubating for up to 3 hours at 37°C, the cells were washed and visualized using a Nikon TE2000 epifluorescence microscope. For in situ dye-transfer studies, 5 μM Cell Tracker Orange was used to label pulmonary endothelial cells in situ and then Calcein Green AM labeled 4T1 cancer cells (without GFP) were administered in the perfusate of mouse lungs and dye transfer to endothelial cells in situ was evaluated by confocal microscopy (BioRad Radiance 2000, Hercules, CA). All fluorescent probes were obtained from Invitrogen Molecular Probes (Eugene, OR).
Immunocytofluorescence for Cx43
Following stable transfection with wild-type or mutant DNA constructs, cells were grown on glass-bottomed 35 mm dishes and were fixed in ice-cold 70% methanol and 20% acetone solution for 10 minutes at 4°C, then rinsed with phosphate buffered saline (PBS) and prepared for immunolabeling. Non-specific antigen sites were blocked with 2% bovine serum albumin (BSA) in PBS, immunolabeled with the Cx43 primary antibody, washed with PBS and labeled with a fluorescent secondary antibody. Cells were then washed in PBS, air-dried, and cover-slipped with a drop of the mounting medium Gold Antifade Reagent containing 4',6-diamidino-2-phenylindole (DAPI) for nuclear staining (Invitrogen, Carlsbad, CA).
Western blot analysis and densitometry
Lysates of 4T1-GFP cells collected from stable colonies after transfection with 30 μg of wild-type or mutant cDNA encoding C61S or G138R were subjected to Western blotting. Blots were immunolabeled for Cx43 using a mouse anti-Cx43 monoclonal antibody (catalog #13-8300, Zymed, San Francisco, CA). Unsaturated blots were scanned and densitometry was performed using Quantity One software (BioRad, Hercules, CA).
Immunohistochemistry was carried out on paraffin-embedded mouse lung tissue sections containing tumor segments. Sections were deparaffinized in two changes of xylene then hydrated in sequential changes of decreasing concentrations of ethanol (100%, 90%, 70%, 50%, 30%) followed by a rinse in distilled water. Antigen retrieval was performed by immersing the slides in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at boiling temperature. Slides were then cooled at room temperature for 20 minutes and rinsed in PBS buffer containing 0.1% Triton X-100. Nonspecific binding was blocked using 5% goat serum and 5% BSA and 0.3% Triton X-100 for 30 minutes. Slides were incubated with mouse anti-Cx43 monoclonal antibody (#35-5000, Zymed, San Francisco, CA) for 2 hours at room temperature. Slides were rinsed again and incubated for 1 hour with a secondary antibody conjugated to a fluorescent probe (Alexa Fluor 647) or horseradish peroxidase (HRP). Slides were then washed in PBS, air-dried, and cover-slipped with a drop of the mounting medium Gold Antifade Reagent containing DAPI for nuclear staining (Invitrogen, Carlsbad, CA).
Quantification of tumor cells adhering to the lung vasculature
Immunohistofluorescence and immunohistochemistry slides were imaged using the Nikon TE2000 epifluorescence microscope with a ×60 objective. Three lungs from animals containing tumors derived from each cell line were used. Four slides from each lung were used for imaging. Twenty contiguous picture frames per slide were acquired. Cells were counted in each stitched meta-picture.
A high-resolution digital fluorescence video imaging system based on a Nikon TE-2000 inverted microscope was used. The imaging system is fitted with two automated 10-position filter wheels for both excitation and emission (Sutter Instruments, model Lambda 10-2), an automated dichroic filter cube changer (Nikon), XYZ axis mechanized stage (Prior Scientific, Inc.), a high-resolution 12-bit Orca-100 ER IEEE1394 digital camera (Hamamatsu Inc.), MetaMorph 7.5 image acquisition, processing, and analysis software with three-dimensional reconstruction and point-spread function-based deconvolution capabilities (Universal Imaging/Molecular Devices). Separately addressable excitation, dichroic, and emission filters allow the maximum flexibility for multiple wavelength and ratio imaging in this system.
For imaging metastatic tumors of GFP-expressing 4T1 cells in situ in the intact mouse lung, an established isolated lung microscopy method was utilized [15, 21]. Some isolated lungs taken from animals with experimental metastases were perfused with Griffonia simplicifolia lectin conjugated with TRITC (Molecular Probes, Eugene, OR) via the pulmonary artery for 30 minutes to label the pulmonary and the tumor microvasculature. The lungs were placed in a specially designed Plexiglas chamber over a coverslip-window at the bottom with the posterior surface of the lung gently touching the coverslip. Subpleural tumors were directly visualized at high magnification (×600) by epifluorescence microscopy using appropriate filters and illumination. Image processing and analysis were performed with MetaMorph software (Molecular Devices, Sunnyvale, CA).
Experimental metastasis model
A syngeneic breast cancer experimental metastasis model was used in the study as described previously . The 4T1-GFP cell line was derived from the 4T1 mouse mammary adenocarcinoma cell line (# CRL-2539, ATCC, Manassas, VA) by stable transfection with eGFP (Clontech, Mountain View, CA). We injected young nu/nu mice (Charles River) into the tail-vein with single-cell suspensions of 4T1-GFP cells at 30 × 103 cells per gram body weight followed by lung isolation, perfusion, and intact organ epifluorescence microscopy up to 21 days after injection. The volume of injected medium with cells was 100 μl. Animals were monitored on a daily basis for signs of respiratory distress. The research was conducted under a protocol reviewed and approved by the University of South Alabama Institutional Animal Care and Use Committee.
Data analysis was performed using SigmaStat (Systat Software Inc., San Jose, CA) using one-way analysis of variance and Bonferroni's test. Data are expressed graphically as means ± standard error of the mean (SEM) using SigmaPlot (Systat Software Inc., San Jose, CA). Differences were considered significant with P < 0.05.
Cx43 expression in tumor cell-endothelial cell co-culture in vitro and in pulmonary metastasis in vivo
Cx43 is upregulated in tumor cell-endothelial cell contact areas
Upregulation of Cx43 marks the sites of micrometastatic foci and metastatic tumor vasculogenesis
Role of Cx43 in tumor cell-endothelial cell interaction in vitro
Cx43 mediates border-sharing and co-growth of 4T1-GFP cells and PMVECs
Cx43 mediates establishment of functional GJIC between 4T1-GFP cells and PMVECs
Immunostaining revealed the presence of Cx43 at the contact area of some tumor cells that were attached to the apical surface of underlying endothelial cells (Figure 10A).
Role of Cx43 in the metastatic efficiency
Cx43 facilitates tumor cell adhesion to lung endothelium
Although a variety of connexins are involved in GJIC in different cell types, the expression of Cx43 in vascular endothelial cells and in many tumor cell lines indicates a primary role for this protein in the heterologous gap junctional communication during tumor cell-endothelial cell interaction [23–28]. Functional GJIC between tumor cells and vascular endothelial cells mediated via Cx43 has been demonstrated [9, 23, 25]. Consequently, it was also shown that quantitative and qualitative changes in connexin expression are associated with tumor progression, proliferation, invasion, and metastasis [9, 25, 29]. Cx43 enhanced the adhesiveness and mediated the invasion in malignant gliomas . In addition, the increased expression of specific connexins correlated with increased invasiveness of lung squamous cell carcinoma or NIH3T3-Ras cells [29, 30] and Cx43 expression was upregulated in urethane-induced mouse lung adenomas . Increased levels of Cx43 mRNA was found in hepatocellular carcinoma tissues  and in the stroma of multiple intestinal neoplasia-mice adenomas . Expression of Cx43 in mammary carcinoma cell lines lacking endogenous Cx43 enabled the formation of heterocellular GJIC with microvascular endothelial cells and increased their diapedesis . Increased cytosolic and plasma membrane expression of Cx43 in lymph node metastases of breast cancer was demonstrated . Cx43 was detectable in normal lung, smaller size tumor, and larger size mouse lung tumors . Our study shows a role for Cx43 in breast cancer metastasis to the lung in a syngeneic, murine experimental metastasis model.
Although connexins are classically considered as gap-junctional proteins, non-junctional roles for connexins in hemichannels with ion-channel-like functions or possessing channel-independent, signaling-type functions have received considerable attention recently [33, 34]. Adhesion-mediated establishment of functional GJIC between lung-metastatic B16F10 melanoma cells and endothelium has been shown to be dependent on the expression of Cx43 in both cell partners .
The marked upregulation of Cx43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, indicates that Cx43 can serve as a marker of micrometastases and tumor vasculogenesis. This raises the possibility of a role for Cx43 in the early incorporation of endothelial cells into pre-hypoxia size tumors as seeds for vasculogenesis.
Cx43 has been shown to enhance angiogenesis in vitro . Tube formation by human umbilical vein endothelial cells cocultured with Cx43-transfected malignant glioma cells or with naturally Cx43-expressing malignant glioma cells was significantly increased compared with tube formation by endothelial cells alone . In addition, upregulation of Cx43 in the hypertrophic myocardium in mice suggested its role in cardiac angiogenesis . These reports are consistent with our finding of upregulation of Cx43 in cancer cell-endothelial cell contact areas. This selective upregulation of Cx43 can be exploited as a marker for sites of intravascular metastases as well as for vasculogenic loci within metastatic tumors. Previously, it had been shown that Cx43 mRNA in normal tissue surrounding lung tumor may act as a molecular marker of nodal micrometastasis in non-small cell lung cancer .
The association between cancer and the lack of communication between cancer cells set the stage for a role of dysfunctional gap junctions in carcinogenesis nearly 40 years ago . Since then, both increased and decreased levels of various connexins shown in different models of primary and metastatic tumors had generated multiple interpretations . The difficulty in developing a unified hypothesis regarding the role of connexins in primary and secondary carcinogenesis may be associated with a multiplicity of contradictory functions that the metastatic cell has to perform during intravasation and distant localization. A recent review predicted that "a gain of function (of gap junctions) may characterize the metastatic stage" . Our results confirm this prediction and we hope that they will set the stage for a renewed assessment of the role of connexins in metastatic tumorigenesis and tumor vasculogenesis.
Cx43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of Cx43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggest that Cx43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis.
This work was supported by NIH P50 AT00428-01 Project 3 (ABA) and the German Research Association (SFB 645, project B2 to KW).
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