Programmable cell adhesion with DNA hybridization is a promising approach for fabricating various tissue architectures without sophisticated instrumentation. However, little is known about how this artificial interaction influences the binding of cell adhesion proteins, E-cadherin. In this work, we designed a planar and fluid lipid membrane displaying E-cadherin and/or single-strand DNA with well-defined densities. Visualization of cells on membranes by fluorescence and interference microscopy revealed cell adhesion to be a two-step process: artificial adhesion by DNA hybridization within a few minutes followed by biological adhesion via cadherin-cadherin binding within hours. Furthermore, we discovered that DNA hybridization can substantially facilitate E-cadherin-mediated cell adhesion. The promotive effect is probably due to the enforced binding between E-cadherin molecules in geometrical confinement between two membranes. Our in vitro model of cell adhesion can potentially be used to design functional synthetic molecules that can regulate cell adhesion via cell adhesion proteins for tissue engineering.
Tissue engineering, which generates functional and complex three-dimensional tissues in vitro, has received considerable attention due to its potential applicability for organ transplantation, disease modeling, and high-throughput drug screening.1–3 In particular, because of the substantial development of stem cell technologies in recent decades, various organlike architectures have been achieved by self-organization processes, including self-assembly, self-patterning, and self-driven morphogenesis.4 However, it is still challenging to reconstitute high-order, complex tissue structures via self-organization alone. To achieve this goal, bioengineering methods such as molecular recognition-based cell assembly5–7 and 3D bioprinting8–10 have emerged as powerful tools to rationally control cell arrangement within tissue architectures. In particular, the control of cell-cell adhesion by the hybridization of synthetic DNA is a promising method for fabricating a wide variety of architectures (i.e., higher-order multicellular structures) without sophisticated instrumentation.6,7 This technique typically utilizes cell membrane modification with single-stranded DNA (ssDNA) that can quickly bind to other cells with complementary sequences. Since an unlimited number of possible coding specificities can be achieved, this DNA technique can potentially provide a high degree of freedom for the construction of tissue architectures with different cell types. However, there is still an open question regarding the influence of artificial cell-cell adhesion via DNA hybridization on the development and maintenance of the resulting tissues. In nature, cells adhere to their surrounding cells and/or extracellular matrix (ECM) via cell adhesion molecules (CAMs).11 The role of CAMs includes not only the physical connection between cells and their environments but also the activation of various intracellular signaling pathways upon cell adhesion. For instance, the intracellular domains of E-cadherin, a well-known cell adhesion protein in vertebrates, form a molecular complex with α/β-catenin, vinculin, and actin fibers,12–14 which activates intracellular signaling pathways for various cellular functions and regulates many important physiological processes, including embryonic development, tumorigenesis, wound healing, and maintenance of the structural integrity of epithelia.15–19 These facts highlight the importance of CAM-mediated cell adhesion in the reconstitution of functional tissues. Thus, to generate functional tissues, it is essential to clarify the influence of the artificially introduced binding (i.e., DNA hybridization) on CAM-mediated cell adhesion, whereas previous studies focused only on demonstrating the fabrication of tissue architectures via DNA hybridization.
In this study, we evaluated the impact of cell surface modification with DNA-polyethylene glycol (PEG)-lipids, ssDNA, and poly(ethylene glycol)-conjugated phospholipid derivatives5 on E-cadherin-mediated cell adhesion [Fig. 1(a)]. For this purpose, we designed a cell-cell adhesion model by seeding living cells onto a planar lipid membrane (PM) displaying E-cadherin and/or DNA-PEG-lipids with well-defined densities [Fig. 1(b)]. Visualization of the cell/PM interfaces by fluorescence and interference microscopy enabled us to distinguish between adhesion sites mediated by E-cadherin and by DNA hybridization. This biophysical approach can provide the first quantitative insights into the mechanism and dynamics of cell-cell adhesion in the presence of artificial and intrinsic interactions.
II. RESULTS AND DISCUSSION
With an average area per SOPC (Alipid) ∼ 65 Å2 and a molar fraction of DGS-NTA (Ni) (xDGS) = 56 mmol. %, the equation yields ⟨dcad⟩ ∼ 34 nm, which translates to ∼860 molecules/μm2. According to the past studies by optical and electron microscopy, the surface density of E-cadherin on living cell membranes was ∼80–500 molecules/μm2,21,22 which is comparable to that on our PMs. We also estimated the average distance between DNA-PEG-lipid molecules, ⟨dDNA⟩ ∼4.1 ± 0.1 nm, by fluorescence intensity-based analysis (see Materials and Methods). These results clearly indicate that cofunctionalization with E-cadherin does not hinder the diffusion of DNA-PEG-lipids on the PM.
By using PMs with well-defied ligand densities, we investigated the adhesion behavior of human breast epithelial cells (MCF-10A) functionalized with DNA-PEG-lipids, which possess complementary DNA sequences to those used for the PMs (TAMRA-labeled polyA20 for cells, and FITC-labeled polyT20 for PMs, respectively). MCF-10A endogenously expresses E-cadherin and thus potentially adheres to the E-cadherin-functionalized PM via homophilic interaction, but the surface density of E-cadherin on the cell membrane was initially low because of treatment with trypsin-EDTA for cell harvesting prior to seeding to PM. It should be also noted that the past literature reported ∼6 h as the time required for the complete recovery of E-cadherin expression in the membrane of MCF-10A cells after trypsin-EDTA treatment.5 Figure 1(d) shows optical microscopy images of MCF-10A cells on PMs (t = 10 min) with three different functionalization conditions: PMs with E-cadherin and DNA-PEG-lipids (PM-cadDNA), DNA-PEG-lipids only (PM-DNA), or E-cadherin only (PM-cad). From the transmission images, the cells showed similar round shapes under all conditions. In contrast, physical contact between cells and PMs [i.e., black regions visualized by reflection interference contrast microscopy (RICM)] was clearly detected when the PM was functionalized with DNA-PEG-lipids. Moreover, confocal fluorescent images showed that the DNA-PEG-lipids on the cell membranes and PMs were colocalized and accumulated at the interface between cells and PMs within 10 min after cell seeding (t = 10 min) [Figs. 1(d) left, 1(d) middle, and 1(e)], while no such accumulation occurred for cells on the PMs without DNA-PEG-lipids [Figs. 1(d) right and 1(f)]. The hybridized DNA-PEG-lipids stably remained at the cell/PM interface at t = 60 min (Fig. S3). These results indicate that DNA hybridization contributes to the formation of the initial physical contact between cells and PMs.
We investigated the cell adhesion dynamics for longer time scales (t ≤ 10 h) to elucidate the impact of DNA-PEG-lipids on the formation of E-cadherin-mediated cell adhesion. Figure 2 shows representative time-course images of cells on PM-cadDNA [Figs. 2(a) and S4(A)], PM-DNA [Figs. 2(b) and S4(B)], and PM-cad [Fig. 2(c)]. In the case of PM-cadDNA, transmission and RICM images show that a cell initially adhered to a PM with a round shape until t ∼ 60 min and gradually began to spread with filopodia on the PM from t ∼ 180 min. Fluorescence images show that the area of the hybridized DNA-PEG-lipids also expanded as the cell spread. Interestingly, we also observed a region where the cell spread without colocalization of hybridized DNA-PEG-lipids [Fig. 2(a)], whereas the cell remained round in PM-DNA conditions even at t = 420 min [Fig. 2(b)]. In addition, the cell on PM-cad showed marginal adhesion at t = 60 min and started to slightly increase its adhesion area at t ∼ 300 min [Fig. 2(c)]. We found that approximately half of the cells could hardly adhere to the PM-cad, indicating weaker adhesion on PM-cad than on PMs with DNA-PEG-lipids. It should be noted that cells did not adhere to a pure SOPC membrane (no surface functionalization) within 600 min (Fig. S5), indicating that the contact regions visualized by RICM were formed by DNA hybridization and/or cadherin-cadherin binding.
|.||PM-cadDNA .||PM-cad .|
|thalf (min)||231.2 ± 23.8||373.1 ± 15.7|
|γ||2.7 ± 0.5||13.8 ± 6.7|
|.||PM-cadDNA .||PM-cad .|
|thalf (min)||231.2 ± 23.8||373.1 ± 15.7|
|γ||2.7 ± 0.5||13.8 ± 6.7|
The cell adhesion dynamics results suggest that cell adhesion to PM-cadDNA involves a two-step process: artificial adhesion via DNA hybridization within a few minutes followed by biological adhesion via cadherin-cadherin binding within hours. The underlying mechanism of this two-step adhesion process can be explained by the difference in reaction kinetics between E-cadherin and DNA-PEG-lipids. Regarding the reaction kinetics of E-cadherin, for simplicity, we here assume two-dimensional adhesion between two different PMs. Wu et al. calculated the two-dimensional molecular kinetic constant KD(2D) (molecules/μm2 as unit), which is analogous to the three-dimensional dissociation constant KD (mol l−1),24 of E-cadherin trans-dimers to be ∼250 molecules/μm2. We found comparable values for the two-dimensional density of E-cadherin on PM (∼860 molecules/μm2) and the surface density of E-cadherin on living cell membranes (∼80–500 molecules/μm2).21,22 Thus, in this density condition, E-cadherin-mediated adhesion between the cell membrane and synthetic PM can be promoted after cell seeding. However, we should also consider that the MCF-10A cells used in this study were treated with trypsin-EDTA prior to seeding, which should decrease the surface density of E-cadherin on the cell membrane during the early time scale. Teramura reported that E-cadherin in the membrane of MCF-10A cells after trypsin-EDTA treatment increases in a sigmoidal manner and requires over 6 h for complete recovery,5 which is consistent with the thalf under PM-cad conditions (∼370 min). This result suggests that the dynamics of cell adhesion on PM-cad was mainly governed by the recovery time scale of E-cadherin expression.
The two-dimensional dissociation constant KD(2D) of DNA-PEG-lipids (A20/T20 base pairs) can be estimated by KD(2D) = hKD, where h is the constrained height. By assuming h = 7 nm (20 base pairs ∼6.8 nm) and KD ∼ 10 pM,25,26 one can obtain KD(2D) ∼4 × 10−5 molecules/μm2, which is 107 times smaller (larger affinity) than KD(2D) of E-cadherin. Considering that DNA-PEG-lipids were densely functionalized on the cell membranes and PMs (∼6 × 104 molecules/μm2), the adhesion between cells and the PM should be completed by DNA hybridization immediately after cell seeding. Indeed, ADNA of both the PM-cadDNA and PM-DNA conditions reached saturation at t = 10 min and became almost constant for 600 min. In addition, as revealed by FRAP experiments, DNA-PEG-lipids are highly mobile in both the fluid PM and PM-cadDNA with diffusion constants of ∼9.3 and 9.4 μm2/s, respectively. Since cell membranes are also fluid (D of non-raft-related fractions on cell membrane > 1 μm2/s at 37 °C),27,28 DNA-PEG-lipids can undergo free diffusion in both cell membranes and PMs. If we use the obtained diffusion constant in PM (D ∼ 9.4 μm2/s) and a typical cell diameter (φ = 10 μm), the time required for the diffusion of DNA-PEG-lipids on the entire cell surface can be calculated as 4π(φ/2)2/D ∼ 134 s, indicating that DNA-PEG-lipids can travel over the entire cell membrane within a few minutes. The results of Figs. 1(e) and S3(A) clearly showed that the accumulation of DNA-PEG-lipids at the cell/PM interface was completed within 10 min. Such fast diffusion together with the high affinity and surface density of DNA-PEG-lipids should lead to the prompt formation of tight contacts between cells and PM via hybridization prior to E-cadherin dimerization.
Considering the above-mentioned difference in reaction kinetics between E-cadherin and DNA-PEG-lipids, it is comprehensible that cell adhesion to PM-cadDNA involves a two-step process. The large difference in γ, the co-operativity coefficient, between PM-cadDNA and PM-cad implies that the dynamics of E-cadherin binding upon cell adhesion is significantly modulated by DNA-PEG-lipids. The obtained half-max moduli, thalf, should represent characteristic time scales for the transition from artificial adhesion via DNA hybridization to biological adhesion via E-cadherin. Interestingly, thalf for PM-cadDNA was approximately half that for PM-cad, which indicates that E-cadherin-mediated cell adhesion in the presence of DNA-PEG-lipids proceeds faster than without DNA-PEG-lipids. This difference in rate may be explained by the biophysical and/or biochemical roles of E-cadherin binding in cell adhesion. As shown in Fig. 3, cells on PM-cadDNA exhibit a substantial area (∼100 μm2) for cell adhesion immediately after cell seeding compared to the case of PM-cad (<10 μm2). In addition, the length of the extracellular domains of DNA-PEG-lipid dimers (polyA20/T20 pair ∼20 nm) is short enough to bring E-cadherin within the length scale of the extracellular domains of E-cadherin dimers (∼40 nm).5,29,30 Therefore, the larger adhesion area mediated by DNA hybridization should provide a larger field for collision and subsequent dimerization between E-cadherin molecules. Furthermore, this induction of E-cadherin binding by DNA hybridization may accelerate the expression of E-cadherin. Some studies have reported that cadherin-cadherin binding can upregulate the expression of E-cadherin via β-catenin or p120-catenin signaling pathways.18,19 Thus, it is plausible that initial contact between cells and PMs mediated by DNA hybridization can promote the binding and subsequent expression of E-cadherin, leading to the faster formation of E-cadherin-mediated cell adhesion on PM-cadDNA than in the absence of DNA-PEG-lipids.
In this work, the effect of artificial cell attachment via DNA hybridization on E-cadherin-mediated cell adhesion was elucidated by using an in vitro model system. Fluid PMs functionalized with a well-defined molecular density of E-cadherin and DNA-PEG-lipids allowed us to quantify the impact of DNA hybridization and E-cadherin binding on the adhesion between cells and PMs. We found cell adhesion to be a two-step process: physical contact mediated by DNA hybridization within minutes and the subsequent E-cadherin-mediated adhesion in hours. Furthermore, we discovered that DNA hybridization can facilitate E-cadherin-mediated cell adhesion. We note that such adhesion promotion via DNA-PEG-lipids may occur with other members of the cadherin family such as N-cadherin because N-cadherin exhibits a similar autoregulation mechanism.31 In addition, the fine-tuned spacing between cell membranes with DNA-PEG lipids should also promote bindings of various cell adhesion molecules. Therefore, we expect that our in vitro model system can be potentially used to design molecular structures that can promote cell adhesion via various cell adhesion proteins, which should become a promising approach in constructing highly ordered, biologically functional tissues using synthetic molecules.
IV. MATERIAL AND METHODS
A. Cell culture
Human breast epithelial cells (MCF-10A) were obtained from the American Type Culture Collection (USA) and routinely cultured in DMEM/F12 (Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12) medium (Thermo Fisher Scientific, USA) containing 10% horse serum (Thermo Fisher Scientific), 20 ng/ml human epidermal growth factor (hEGF, Pepro Tech, USA), 0.5 μg/ml hydrocortisone (Sigma-Aldrich, USA), 20 μg/ml insulin (Sigma-Aldrich), and 50 unit/ml penicillin-streptomycin (Thermo Fisher Scientific). Cells were maintained in an incubator at 37 °C with 5% CO2 supply.
B. Preparation of small unilamellar vesicle (SUV)
SOPC and DGS-NTA (Ni) lipid powders were purchased from Avanti Polar Lipid (USA) and used without further purification. SOPC or DGS-NTA (Ni) powder was dissolved into chloroform and stored at −20 °C as stock solutions [10 mg/ml for SOPC and 0.1 mg/ml for DGS-NTA (Ni), respectively]. To prepare SUV, 200 μl of SOPC and 1.5 μl of DGS-NTA (Ni) from the stock solutions were mixed in a glass vial, which was cleaned by the modified RCA protocol beforehand.20,32 The molar ratio of DGS-NTA (Ni) can be estimated to be 56 mmol. % with respect to SOPC. The chloroform in the mixture was evaporated under gentle nitrogen gas flow to form a dry lipid film and then stored in a vacuum chamber overnight to remove the residual solvent. Next, a dry lipid film was suspended in 4 ml of HEPES-buffered saline (HBS, 10 mM HEPES, 150 mM NaCl, pH 7.4) and incubated for 1 h at 37 °C. The lipid suspensions were then sonicated by using a tip sonicator (Q55, Qsonica, USA) for 60 min. Impurities (mostly fragments of the titanium tip of the sonicator) were precipitated by centrifugation (14 800 rpm for 10 min), and the supernatants were collected as the SUV solution. The final concentration of SOPC in the solution was 0.5 mg/ml. In this study, we also prepared SUV solution containing 0.5 mg/ml SOPC + 0.5 mol. % Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Texas Red-DHPE, Thermo-Fisher Scientific) to confirm the deposition of PMs on glass substrates as well as their diffusivity.
C. Preparation of PM and its functionalization with E-cadherin and/or DNA-PEG-lipid
Poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning Toray, Japan) and a coverslip (Matsunami Glass, Japan) were used to prepare a chamber for this experiment. A silicone prepolymer mixed with a cross-linker at a ratio of 10:1 (in volume) was baked for more than 3 h at 80 °C. Afterwards, PDMS was hollowed out by a punch with a diameter of 6 mm. The coverslip was cleaned and hydrophilized by the modified RCA protocol, followed by treatment with an UV/Ozone cleaner (ProCleaner Plus, BioForce Nanoscience, USA). Finally, the coverslip was fixed together with the hollowed PDMS, which was also treated with O2 plasma, to form the chamber. PMs were prepared by the deposition of 100 μl of SUV solution in the chamber for 30 min at room temperature. After the removal of unbound lipids by washing with HBS, 10 mM NiCl2 solution was added to the chamber (final conc. 1 mM) and incubated for 30 min at room temperature. The chamber was again washed with HBS, and 8 μl of 250 μg/ml histidine-tagged E-cadherin (R&D Systems, USA) was added and incubated for 90 min at room temperature, which allows for the uniform functionalization of a planar membrane with E-cadherin via the Ni2+ chelation.20,33 Thereafter, the chamber was washed again with HBS, and 4 μl of 26 μg/ml FITC-labeled polyT20-PEG-lipid solution, which was synthesized as previously described,5,34 was added and incubated for 30 min at 37 °C. Finally, the PM was rinsed with L15 medium (20 mM HEPES buffered, Thermo-Fisher Scientific) to remove excess components. In the case of the FRAP measurement of E-cadherin (Fig. S1), fluorescently labeled E-cadherin was prepared by mixing a 6-Carboxytetramethylrhodamine, Succinimidyl Ester (TAMRA-SE, Thermo-Fisher Scientific) according to the manufacture's protocol. Then the TAMRA-labeled E-cadherin was anchored on a PM with 5.6 mol. % of DGS-NTA under the same procedure as mentioned above.
D. Modification of the cell membrane with DNA-PEG-lipid
Cultured cells were harvested with trypsin-EDTA similar to past works of tissue engineering with DNA-PEG lipids5,35 and then collected by centrifugation for 3 min at 1000 rpm. After removal of the supernatants, the cell pellets were incubated with 30 μl of 260 μg/ml TAMRA-labeled polyA20-PEG-lipid solution for 30 min at 37 °C. Cells were then washed and resuspended in L15 medium and seeded onto the PM in the chamber.
F. Image analysis
G. FRAP measurement
H. Determination of the mean distance between DNA-PEG-lipids
The mean distance between DNA-PEG-lipids within PMs was calculated from the concentration of unbound DNA-PEG-lipids. First, fluorescence spectra of HBS solutions containing FITC-labeled DNA-PEG-lipids with known concentrations (0.5–2 μg/ml) were obtained by using a fluorescence spectrometer (FP-6300, JASCO, Japan) [Fig. S8(A)]. The excitation wavelength was 495 nm. Then, a calibration curve was obtained by plotting the fluorescence intensities at 520 nm as a function of the concentration of DNA-PEG-lipids [Fig. S8(B)]. Next, PMs with E-cadherin were prepared in a PDMS chamber with a hollow diameter of 20 mm, which is larger than those used in the cell experiments. The PM was incubated with 1 ml of a solution containing 1.5 μg/ml FITC-labeled DNA-PEG-lipids for 30 min at 37 °C. The solution was then diluted to 2 ml. After gentle pipetting, 1 ml of the solution was collected, and the fluorescence spectrum was measured by using a fluorescence spectrometer. The concentration of unbound DNA-PEG-lipids was calculated from the calibration curve. Finally, by taking the molecular weight of DNA-PEG-lipids (5000) and the known chamber area, a mean distance between DNA-PEG-lipids ⟨dDNA⟩ could be determined. We conducted three independent experiments to determine the mean distance.
I. Ethics approval
Ethics approval is not required to carry out this work.
See the supplementary material for FRAP results of PMs functionalized with TAMRA-labeled E-cadherins, FRAP results of PMs functionalized with FITC-labeled DNA-PEG-lipids or Texas red-DHPE (no DNA-PEG-lipids), confocal images of a cell with or without DNA-PEG-lipids at t = 60 min, lower magnification images of the adhesion dynamics of cells on PM-cadDNA and PM-DNA, images of the adhesion dynamics of cells on a pure lipid membrane, scheme of cell adhesion area analysis conducted using ImageJ software, time-course histograms of adhesion area, and quantification of DNA-PEG-lipids on PMs.
This work was partly supported by grants from the Japan Society for the Promotion of Science (JSPS) KAKENHI (Nos. 15H05351, 16K12868, and 23106005) and Takeda Science Foundation.
The authors declare no conflicts of interest associated with this manuscript.