1 Materials and methods

1.1 Materials and reagents

The biochemical materials used in this study include acetone, biotin-conjugated BSA (Bioss Antibodies, Beijing, China), PBS (Thermo Scientific, Waltham, MA, USA), Streptavidin (Sigma, Merck KGaA, Darmstadt, Germany), biotin-conjugated concanavalin A (Sigma,Merck KGaA, Darmstadt, Germany), 3-Aminopropy triethoxysilane(Sigma, Merck KGaA, Darmstadt, Germany), 25% glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd. Shanghai, China), Rabbit-anti-human CD20 (Bioss Antibodies, Beijing, China), FITC-conjugated horse-anti-rabbit secondary antibody (Bioss Antibodies, Beijing, China), CFDA SE (Beyotime, Shanghai, China), and poly-L-lysine (Solarbio, Beijing, China). The AFM probe type used here is MLCT-O10 (Bruker, Santa Barbara, CA, USA). Rituximab was provided by the Affiliated Hospital of Military Science Academy of the PLA (Beijing, China).

1.2 Cell culture and preparation

B lymphoma cells were obtained from Burkitt lymphoma Raji cell lines which were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Raji cells were cultured in RPMI 1640 medium (Thermo Scientific, Waltham, MA, USA) containing 10% fetal bovine serum at 37 ℃ (5% CO₂). The 2 ml Raji cell suspension was centrifuged at the speed of 1000 r/min for 10 min. After centrifugation, the supernatant was removed. Then 1 ml PBS was added into the tube to resuspend the Raji cells.

1.3 Functionalization of AFM cantilevers

According to the published protocol^[23], the tipless cantilevers with a nominal spring constant of 0.01 N/m were coated with concanavalin A. The tipless cantilevers were firstly cleaned by UV-radiation for 45 min. Then the cantilevers were incubated in 50 μl of biotin-conjugated bovine serum albumin (BSA) (0.5 g/L solution in NaHCO₃) at 37 ℃ overnight. After incubation, the cantilevers were washed with PBS for three times to remove the unbounded molecules and then incubated in 50 μl of streptavidin (0.5 g/L solution in PBS) for 30 min. After washing the cantilevers with PBS for three times, the cantilevers were incubated in 50 μl of biotin-conjugated concanavalin A (0.4 g/L solution in PBS) for 30 min. After incubation, the cantilevers were washed with PBS for three times.

1.4 Substrate functionalization

Rituximab molecules were coated on the substrate of Petri dishes whose diameter was 60 mm. The substrate of the Petri dish was divided into several areas with the use of a marker pen, which facilitated us to coat each area of the substrate with different concentrations of rituximab. The dish was firstly silanized by 2% APTES for 10 min. After silanization, the dish was rinsed with pure water. Then the dish was treated by 0.5% glutaraldehyde for 30 min. After washing the dish with pure water, the six regions at the substrate of the dish were incubated with different concentrations of rituximab for 30 min. For control, the substrate was incubated with BSA. After incubation, the dish was washed by PBS and then free PBS was added into the substrate to keep the activities of the molecules coated on the substrate of the dish.

Fig. 1 Fluorescence microscopy experiments verifying substrate functionalization

NOTE: (a) Optical bright field image of the substrate of the Petri dish. (b) Fluorescence image of the substrate of the Petri dish without rituximab functionalization. (c) Fluorescence image of the substrate of the Petri dish coated with rituximab.

In order to examine whether rituximab had been coated on the substrate of Petri dish, fluorescence microscopy experiments were performed. The Petri dish coated with rituximab was firstly incubated with rabbit-anti-human CD20 antibody solution for 30 min at 37 ℃. After incubation, the Petri dish was washed by PBS for three times. Then the substrate was incubated with FITC-conjugated horse-anti-rabbit secondary antibody for 30 min. After incubation, the Petri dish was washed by PBS for three times, and then fluorescence images were recorded. For control, Petri dish without rituximab functionalization was also observed. As shown in Figure ,1, no fluorescence was observed for the Petri dish without rituximab functionalization, while the Petri dish with rituximab functionalization exhibited bright fluorescence, indicating that rituximab had been coated on the substrate of the Petri dish.

1.5 Single-cell probe preparation

The single-cell AFM probe was prepared by attaching single cells onto the concanavalin A-coated AFM tipless cantilever based on AFM micromanipulations with the assistance of optical microscopy (Catalyst AFM, Bruker, Santa Barbara, CA, USA), as shown in Figure 2. The Raji cell suspension was added into the sub-area of the substrate of the Petri dish (this sub-area was not coated by rituximab). The cells deposited onto the substrate in a few minutes. Then the concanavalin A-functionalized tipless cantilever was moved to one Raji cell under the guidance of optical microscopy (Figure 2a). Next, the cantilever was controlled to gradually approach and contact the cell for 30 s with a contact force 4 nN (Figure 2b). After that, the cantilever retracted from the substrate and the cell was attached to the AFM cantilever to form single-cell probe (Figure 2c).

Fig. 2 Preparation and activity verification of single-cell AFM probe

NOTE: (a-c) Schematic and optical images of preparing single-cell probe. The optical images are under the schematic diagrams. (a) The concanavalin A-coated cantilever was approaching a single Raji cell deposited on the substrate. (b) The cantilever contacted the cell and dwelt for 30 s to allow the binding of cell to the concanavalin A on the cantilever. (c) The cantilever retracted from the substrate and the cell was attached to the cantilever to form single-cell probe. (d) The single-cell probe was stained with CFDA SE and the fluorescence indicated that the cell on the cantilever was alive.

In order to examine the activities of the single-cell probe, the cell attached to the AFM probe was stained with CFDA SE. The AFM single-cell probe was placed in 1 ml CFDA SE working solution and then incubated at 37 ℃ for 15 min. After incubation, the probe was washed by PBS for three times. Figure 2d shows the fluorescence image of the single-cell probe stained by CFDA SE. The cell attached to the cantilever strikingly exhibited green fluorescence, indicating that the cell attached onto the probe was alive.

1.6 Single-cell force spectroscopy

The procedure of using single-cell force spectroscopy to measure the adhesion forces between Raji cells and rituximab-coated substrate was shown in Figure 3. Firstly, the single-cell probe was moved to gradually approach and contact the rituximab-coated substrate with a constant loading force (the loading force is adjusted by changing the trigger threshold of force ramp in the user interface of AFM nanomanipulation software). The single-cell probe dwelt on the substrate for a period of time and then retracted from the substrate. During the retract process, the rupture between cells on the cantilever and rituximab on the substrate resulted in the specific force peaks in the force curve. The magnitude of the force peak corresponded to the cellular detachment force. Figure 3a shows the optical image of measurements and Figure 3b shows the recorded representative force curves. We can clearly see the force peaks in the retract curve, which indicates the CD20-rituximab unbinding events. In some cases, multiple molecular unbinding events were observed from the force curve, which exhibited stepped peaks (top curve in Figure 3b). In some cases, single unbinding events were observed, which exhibited individual peaks (bottom curve in Figure 3b). For each sub-area of the substrate coated with different concentrations of rituximab, 100 force curves were recorded. For control, the substrate coated with BSA was also used for measurements.

Fig. 3 Measuring the adhesion force between Raji cells and rituximab-coated substrate using single-cell force spectroscopy

NOTE: (a) Optical images of controlling single-cell probe to perform approach-dwell-retract movements on the rituximab-coated substrate. (b) Representative force-distance curves recorded during the measurements. Force curves were recorded with contact time 1 s and loading force 1 nN. The measured detachment force is 0.38 nN for the top force curve and 0.15 nN for the bottom force curve respectively.

2 Results and discussion

2.1 Detecting the molecular adhesion force between Raji cells and BSA-coated substrate

Fig. 4 The adhesion forces between Raji cells and BSA-coated substrate measured by AFM

NOTE: (a) Cellular adhesion forces measured by varying the contact times between cell and substrate. (b) Cellular adhesion forces measured by varying the loading forces. (c) A typical force curve recorded during the measurements.

We firstly measured the adhesion force between Raji cells and BSA-coated substrate for control experiments, as shown in Figure 4. The substrate of the Petri dish was coated by 50 mg/L BSA. Figure 4c is a typical force curves which clearly shows the force peak in the retract curve, indicating the adhesion between Raji cells and BSA. Figure 4a shows the results of the adhesion forces measured at different contact times between cell and substrate. We can see that the cellular adhesion force increased as the increase of contact times. However, when the contact time was larger than 2 s, the adhesion force measured by AFM largely kept stable (~ 0.35 nN) even the contact time further increased. Figure 4b shows the results of the adhesion forces measured at different loading forces between cell and substrate. We can see that on the whole the influence of loading force on the measured cellular adhesion force was weak and the cellular adhesion force kept stable when varying the loading forces.

2.2 Detecting the molecular adhesion force between Raji cells and rituximab-coated substrate

Figure 5 shows the adhesion force between Raji cells and rituximab-coated substrate measured with different concentrations of rituximab. Six concentrations of rituximab (0 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L, 100 mg/L) were used for coating the different sub-regions of the substrate of the Petri dish. The measurement of contact time is 1 s, and the trigger threshold is 1 nN. For each sub-region of the substrate, 100 force curves were recorded at 5 μm×5 μm areas. We can see that the adhesion force significantly increased from 0.145 nN to 0.699 nN when the concentration of rituximab increased from 0 mg/L to 60 mg/L. When the concentration of rituximab further increased, the adhesion force basically kept unchanged.

Fig. 5 The adhesion forces between Raji cells and rituximab-coated substrate measured by varying the concentrations of rituximab coated on the substrate

Figure 6 shows the adhesion force between Raji cells and rituximab-coated substrate measured with different contact times. The results of Figure 5 have shown that the suitable concentration of rituximab coated on the substrate for measuring adhesion force was 60 mg/L, when the trigger threshold is 1 nN. Then we measured the adhesion force on the substrate coated by 60 mg/L rituximab by varying the contact times between cell and substrate. As shown in Figure ,6, eight different contact times (0.1 s, 0.5 s, 1 s, 1.5 s, 2 s, 2.5 s, 3 s, 4 s) were used for the measurements. When the contact time increased from 0.1 s to 1 s, the adhesion force rapidly increased from 0.367 nN to 0.668 nN. When the contact time increased to 2.5 s, the adhesion force slowly increased to about 0.763 nN and then kept stable.

Fig. 6 Adhesion forces between Raji cells and rituximab-coated substrate measured by varying the contact times between cell probe and substrate

NOTE: The substrate was coated by 60 mg/L rituximab.

Figure 7 shows the adhesion force between Raji cells and rituximab-coated substrate measured with different loading forces of AFM probe. According to the results in Figure ,6, we used the contact time 1 s for measurements. From Figure ,7, we can see that the adhesion forces nearly kept stable (~ 0.6 nN) when the loading force increased from 0.1 nN to 3 nN, indicating that the influence of loading force of AFM on the measurements of adhesion forces was weak.

Fig. 7 The adhesion forces between Raji cells and rituximab-coated substrate measured by varying the loading forces

NOTE: The substrate was coated by 60 mg/L rituximab.

Comparing the adhesion forces between Raji-BSA and Raji-rituximab, we can see that the adhesion forces between Raji cells and rituximab (~ 0.6 nN, Figure 7) were significantly larger than the adhesion forces between Raji cells and BSA (~ 0.35 nN, Figure 4). This is due to the different types of molecular interactions involved in these two types of adhesion. Rituximab specifically binds to the CD20 antigen on the Raji cell, which activates the signaling pathways for killing Raji cells^[24], whereas the binding of Raji-BSA was due to the unspecific molecular interactions^[25]. Therefore, the specific molecular binding of Raji-rituximab and the unspecific binding of Raji-BSA results in the different adhesion forces.

2.3 Detecting the cellular adhesion force between Raji cells

Figure 8 shows the adhesion forces between Raji cells. With the use of poly-L-lysine, single living lymphoma cells can be immobilized on the substrate^[26]. By coating the substrate of Petri dish with poly-L-lysine, Raji cells were immobilized on the substrate. Under the guidance of optical microscopy, the single-cell probe was moved to a Raji cell (Figure 8a) and contacted the cell for the AFM-based SCFS measurements (Figure 8b). Adhesion forces between Raji cells were obtained by analyzing the recorded force curves. Figure 8c shows the adhesion forces measured by changing the contact times. We can see that the adhesion force increased from 0.161 nN to 0.457 nN when the contact time increased from 0.1 s to 4 s. Figure 8d shows the adhesion forces measured by changing the loading force of AFM probe. We can see that the variations of the loading force of AFM probe did not cause the significant changes of the adhesion forces between Raji cells. The results of Figure 8 showed the adhesive interactions between B lymphoma cells. On the surface of B-cell lymphoma cells, there are many different types of adhesion molecules, such as CD44 and CD24^[27]. These adhesion molecules regulate the binding of Raji cells to perform various biological functions, which may cause the adhesive interactions between Raji cells. SCFS has been widely used to investigate cell adhesion, but these studies are commonly performed on adherent cells^[28]. So far the information about the adhesive behaviors of single lymphoma cells is still scarce. Here, taking lymphoma cells as an example, our results prove the capabilities of AFM in detecting the cellular adhesive behaviors between human suspended cells, which will benefit the investigations of cell adhesion involved in the physiological and pathological changes of human suspended cells.

Fig. 8 The adhesion forces between Raji cells

NOTE: One Raji cell was attached to AFM cantilever and one Raji cell was immobilized on the substrate. The adhesion forces between the two Raji cells were measured by AFM single-cell force spectroscopy. (a) The single-cell probe gradually approached individual Raji cell. (b) The single-cell probe contacted the Raji cell to perform measurements. (c) The adhesion forces between Raji cells measured by varying the contact times. (d) The adhesion forces between Raji cells measured by varying the loading forces.

AFM-based SCFS provides a powerful tool for investigating molecular and cellular adhesive interactions. Traditionally, AFM measures molecular interactions by linking receptors onto AFM tip and then performing single-molecule force spectroscopy on cells^[29,30]. The receptor-ligand interactions on the cell surface can then be probed. However, a disadvantage of this method is that it requires the biochemical functionalization of AFM tip, which is quite complex and time-consuming. Here, we directly attached single cell onto AFM cantilever and coated antibodies on the substrate, which allowed measurements of antigen-antibody molecular interactions. The method presented here was simple compared with traditional single-molecule force spectroscopy, thus providing a novel idea for investigating molecular interactions by AFM. Besides, we have expanded AFM-based SCFS to human suspended cells (lymphoma Raji cells) and the results showed the adhesive interactions between lymphoma cells. Detailed procedures were shown here to measure the molecular and cellular adhesion forces by AFM, including substrate treatment, cell-probe preparation, force measurements and analysis. The methods can be utilized to investigate the adhesive behaviors of other types of suspended cells, which will facilitate the studies of cell mechanics in tumor development and progression.

In summary, this work has demonstrated the use of AFM-based SCFS to quantitatively measure the molecular and cellular adhesive interactions taking place in lymphoma cells, which will potentially benefit the biomechanical studies for understanding the cell adhesion in cancer.

References