Promotion and Detection of Cell–Cell Interactions through a Bioorthogonal Approach

Manipulation of cell–cell interactions via cell surface modification is crucial in tissue engineering and cell-based therapy. To be able to monitor intercellular interactions, it can also provide useful information for understanding how the cells interact and communicate. We report herein a facile bioorthogonal strategy to promote and monitor cell–cell interactions. It involves the use of a maleimide-appended tetrazine-caged boron dipyrromethene (BODIPY)-based fluorescent probe and a maleimide-substituted bicyclo[6.1.0]non-4-yne (BCN) to modify the membrane of macrophage (RAW 264.7) and cancer (HT29, HeLa, and A431) cells, respectively, via maleimide–thiol conjugation. After modification, the two kinds of cells interact strongly through inverse electron-demand Diels–Alder reaction of the surface tetrazine and BCN moieties. The coupling also disrupts the tetrazine quenching unit, restoring the fluorescence emission of the BODIPY core on the cell–cell interface, and promotes phagocytosis. Hence, this approach can promote and facilitate the detection of intercellular interactions, rendering it potentially useful for macrophage-based immunotherapy.


General
All the reactions were performed under an atmosphere of nitrogen.DMF and CH2Cl2 were dried using an INERT solvent purification system.All other solvents and reagents were used as received.Chromatographic purification was performed on silica gel (Macherey-Nagel 230-400 mesh) with the indicated eluent.Compounds 4 R1 and 8 R2 and the GE11 peptide R3 were prepared as described.
Electrospray ionization (ESI) mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer.SEM images were obtained using a Hitachi SU8010 cold-field scanning electron microscope.Steady-state fluorescence spectra were taken on a Horiba FluoroMax spectrofluorometer.
Reverse-phase high-performance liquid chromatography (HPLC) separation was performed on an Apollo-C18 column (5 μm, 4.6 mm × 150 mm) at a flow rate of 1 mL min -1 for analytical purpose or on a XBridge BEH300 Prep C18 column (5 μm, 10 mm × 250 mm) at a flow rate of 3 mL min -1 for preparative purpose, using a Waters system equipped with a Waters 1525 binary pump and a Waters 2998 photodiode array detector.The solvents used for HPLC analysis were of HPLC grade.The HPLC conditions were set as follows: solvent A = S6 0.1% TFA in acetonitrile; solvent B = 0.1% TFA in distilled water.The gradient was 100% B in the first 5 min, and then changed to 100% A in 35 min, maintained under this condition for 10 min, changed back to 100% B in 5 min, and finally kept under this condition for 10 min.

Preparation of 3
Resin 1 with the peptide sequence EEHRPEGGGGSK was synthesized manually using a modified Fmoc SPPS protocol with the commercially available N-α-Fmoc-protected amino acids as starting materials.The rink amide resin was used as the solid support, and HATU was used as the carboxyl group activating agent.The swelled resin was treated with piperidine (20%) in DMF for 20 min twice before being coupled with the first amino acid.The Fmoc-protected amino acid (4 equiv.),HATU (4 equiv.), and DIPEA (8 equiv.)were used for each coupling.
After the final coupling, the N-terminal of the peptide resin was coupled with 6maleimidohexanoic acid (2) (4 equiv.)overnight in the presence of HATU (4 equiv.)and DIPEA (8 equiv.).After being washed with DMF and CH2Cl2 twice, the peptide resin was treated with a mixture of TFA (95%), TIPS (2.5%), and H2O (2.5%) (4 mL) for 2 h to detach the peptide from the resin and remove the protecting groups.The resin was removed by filtration and the filtrate was precipitated by the addition of cold diethyl ether.After centrifugation, the supernatant was removed.The white solid obtained was re-dissolved in DMF (0.5 mL) and then precipitated again by the addition of diethyl ether.This purification procedure was repeated twice, and then the product was further purified using HPLC by monitoring the absorbance at 220 nm.The HPLC yield was determined to be 96%, and the S7 purity of 3 was found to be >95% by HPLC analysis.MS (MALDI-TOF): m/z calcd for C59H90N19O23 [M+H] + 1432.645,found 1432.639.

Preparation of 9
To a 100 mL Schlenk tube equipped with a stirrer bar, 7 (7.0 g, 28.3 mmol), 8 (1.0 g, 5.6 mmol), Zn(OTf)2 (1.0 g, 2.8 mmol), and NH2NH2•H2O (14.1 g, 0.28 mol) were added.The vessel was sealed, and the mixture was stirred in an oil bath at 60 °C for 24 h.The mixture was then cooled to room temperature, and the seal was removed.A solution of NaNO2 (7.6 g, 0.11 mol) in water (20 mL) was slowly added to the mixture, followed by slow addition of 1 M HCl, during which the mixture turned to bright red in color with gas evolved.Addition of 1 M HCl continued until the gas evolution ceased and the pH value reached ca. 3. The mixture was extracted with ethyl acetate (50 mL x 2).The combined organic phase was dried over anhydrous Na2SO4 and then evaporated to dryness under reduced pressure.The residue was purified by chromatography on silica gel with CHCl3/MeOH (50:1, v/v) as the eluent to afford 9 as a red solid (0.82 g, 32%).

Fluorescence Analysis of the Activation of Mal-TzBDP by BCN-OH
The bioorthogonal reaction was performed in a 1 cm × 1 cm quartz cuvette.A stock solution of Mal-TzBDP in DMF (2 mM) was first prepared, which was diluted with PBS at pH 7.4 to give a 2 µM solution.Another stock solution of BCN-OH in DMF (20 mM) was also prepared.
An aliquot of this solution was then added to the PBS solution of Mal-TzBDP prepared above to make the final concentration of BCN-OH at 10 µM.The fluorescence spectrum of the mixture was recorded from 500 to 650 nm over a period of 60 min upon excitation at 488 nm.

LC-MS Analysis of the Activation of Mal-TzBDP by BCN-OH
acid (0.4 mL).The activity of trypsin was quenched with the culture medium (0.5 mL), and the mixture was centrifuged at 1500 rpm for 3 min.The pellet was washed with PBS (1 mL) three times and then centrifuged.The cells were then suspended in HBSS (1 mL) and subjected to flow cytometric analysis using a BD FACSVerse flow cytometer (Becton Dickinson) with 10 4 cells counted in each sample.Cell fragments were excluded with forward and side-scatter gating to ensure that all the detected signals were originated from relatively intact cells.Signals from the activated Mal-TzBDP were recorded in Chanel-FITC.All experiments were performed in triplicate.The results were compared with those without the post-incubation with GE11-BCN.

Cell Surface Modification with Mal-BCN
Approximately 2 × 10 5 A431 cells in 2 mL of the culture medium were incubated on confocal dishes of 35 mm diameter at 37 °C with 5% CO2 overnight.After removing the medium, the cells were rinsed with PBS and then incubated with 1 mM of TCEP for 30 min.After that, the cells were rinsed with PBS twice with or without further incubation with Mal-BCN (10 µM) for 30 min.To verify whether Mal-BCN had been immobilized on the cell surface, the cells, after being rinsed with PBS twice, were further incubated with Mal-TzBDP (4 µM) for 30 min.
The cells were rinsed with PBS again and then immersed in HBSS (1 mL) for confocal microscopic and flow cytometric studies as described above.

Study of Photocytotoxicity
Approximately 1 × 10 4 HT29, A431, and RAW 264.7 cells per well in the culture medium were S15 inoculated in 96-well plates and incubated at 37 o C with 5% CO2 overnight.The cells were first treated with TCEP (1 mM) for 30 min.After being rinsed with PBS for three times, the cells were further incubated with Mal-TzBDP or Mal-BCN at different concentrations for 30 min.
The cells were then rinsed with PBS for three times and refed with 100 µL of the culture medium.Cell viability was determined by means of a colorimetric MTT assay as described previously.R4

Click-Induced Cell-Cell Interactions
A431 and HT29 cells at a density of 2 × 10 6 cells mL -1 were suspended in a CellTracker Red CMTPX Dye (2 µM) solution for 30 min.After being rinsed with PBS twice, the cells were further modified with Mal-BCN (10, 20, or 40 µM for A431 cells; 40 µM for HT29 cells) as described above.Similarly, RAW 264.7 cells at a density of 2 × 10 6 cells mL -1 were suspended in a CellTrace Violet (4 µM) solution for 30 min and then modified with Mal-TzBDP (8 µM) as described above.The native and Mal-BCN-modified A431 or HT29 cells were then cocultured with the Mal-TzBDP-modified RAW 264.7 cells in a 1:1 or 1:3 ratio.The cell mixtures were shaken for 30 min at 37 °C in a thermostatic oscillator.The cells were then suspended in the culture medium and examined with a Leica TCS SP8 high-speed confocal microscope.Lasers at 405, 488, and 532 nm were used to excited CellTrace Violet, the activated Mal-TzBDP, and CellTracker Red CMTPX Dye, respectively.The images were digitized and analyzed using a Leica Application Suite X software.For quantification of the cell-cell connecting efficiency, flow cytometry was used to determine the percentages of discrete and assembled cells.All the assembled systems were diluted to fourfold series with S16 PBS.Cell fragments were excluded with forward and side-scatter gating to ensure that all the detected signals were originated from relatively intact cells.Signals from CellTrace Violet were recorded in Chanel V-450A, while signals from CellTracke Red CMTPX Dye were recorded in Chanel APC-A.The flow cytometry diagrams presented were obtained from a population of 1 × 10 4 cells.

Time-Lapsed Confocal Imaging of Phagocytosis
The A431 cells, which had been treated with 40 µM of Mal-BCN, and the Mal-TzBDPmodified RAW 264.7 cells were co-cultured in 1:1 ratio for 30 min as described above.The cell mixture was then incubated in the culture medium for a further 12 h, and then the cell-cell interactions were monitored with a Leica TCS SP8 high-speed confocal microscope over a period of 12 h.Time-lapsed images were captured at every 15 min interval.

SEM Measurements
After the co-culturing of A431* and RAW 264.7* cells (1:1) for 30 min as described above, the cells were incubated on clean glass slices at 37 °C with 5% CO2 for 12, 24, and 48 h, respectively.The medium was then removed.The cells, after being rinsed with Sorensen's phosphate buffer (SPB) (0.1 M at pH 7.2), were immediately fixed with a 2.5% glutaraldehyde solution in SPB at room temperature for 30 min.The cells were then rinsed with SPB for 3 times (10 min each) and fixed in 1% osmium tetroxide for 30 min.After that, the cells were washed with distilled water for 3 times (10 min each) and then dehydrated with a graded ethanol series: 80% (10 min), 90% (10 min), 95% (15 min) for two times, and finally 100% (15 min) S17 for two times, before being dried in a critical point dryer.The specimens were mounted on specimen stubs and then coated with gold-palladium on a sputter coater before being examined under a scanning electron microscope.

Study of Phagocytosis Efficiency of Activated RAW 264.7*
RAW 264.7 cells were first treated with lipopolysaccharide (LPS) at a concentration of 100 ng mL -1 for 24 h to activate their phagocytic activity.After being rinsed with PBS twice, the cells at a density of 2 × 10 6 cells mL -1 were suspended in a CellTrace Violet (4 µM) solution for 30 min and then modified with Mal-TzBDP (8 µM) as described above.Similarly, A431 cells at a density of 2 × 10 6 cells mL -1 were suspended in a CellTracker Red CMTPX Dye (2 µM) solution for 30 min and then modified with Mal-BCN (40 µM) as described above.
Subsequently, the modified RAW 264.7* cells were co-cultured with A431* cells or the unmodified A431 cells in a 1:1 ratio for 30 min, followed by further incubation in the culture medium for a further 24 h.The phagocytosis of A431*/A431 cells by RAW 264.7* was monitored with a Leica TCS SP8 high-speed confocal microscope.

Figure S1
Figure S1 Change in fluorescence spectrum of Mal-TzBDP (2 µM) in the (a) absence and

Figure S2
Figure S2 Bright field, fluorescence, and the merged confocal images of HT29 and HeLa

Figure
Figure S3 (a) Bright field, fluorescence, and the merged confocal images of HeLa cells after

Figure S4
Figure S4 Bright field, fluorescence, and the merged confocal images of A431 cells after

Figure
Figure S5 (a) Confocal images of HT29 and RAW 264.7 cells after sequential incubation

Figure S6
Figure S6Cell viability of HT29, A431, and RAW 264.7 cells after incubation with TCEP

Figure
Figure S7 (a) Cell viability of RAW 264.7 cells after sequential incubation with TCEP (1

Figure S8
Figure S8Flow cytometric analysis of different cell assemblies: (a) For native A431 or

Figure S9
Figure S9 Confocal images of the cell assemblies of A431* (treated with 40 μM of Mal-

Figure 10
Figure 10 Confocal images of the cell assemblies of A431* (treated with 40 μM of Mal-

Figure S2 .Figure S3 .
Figure S2.Bright field, fluorescence, and the merged confocal images of HT29 and HeLa cells