In Situ Monitoring of Fluid Shear Stress Enhanced Adherence of

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In situ monitoring of fluid shear stress enhanced adherence of bacteria to cancer cells on microfluidic chip Wanling Zhang, Sifeng Mao, Ziyi He, Zengnan Wu, and Jin-Ming Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00394 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 8, 2019

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Analytical Chemistry

In situ monitoring of fluid shear stress enhanced adherence of bacteria to cancer cells on microfluidic chip Wanling Zhang, Sifeng Mao, Ziyi He, Zengnan Wu, Jin-Ming Lin* Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China ABSTRACT: Mechanosensing mechanisms for surface recognition by bacteria play an important role in inflammation and phagocytosis. Here, we describe a set of DNA probes for revealing microbe adherence to cancer cells under fluid shear stress. DNA probes modified with a biotin group, an azido group, hexadecanoic acid were indiscriminately anchored to cell surface, acting as indicators for the membrane proteins, cell-surface carbohydrate, and phospholipids. When cancer cells were exposed to bacteria in fluid, accumulation of membrane proteins enhanced indicated by the strong fluorescence aggregation, meanwhile the accumulation of cell-surface carbohydrate and phospholipids weakened indicated by attenuated fluorescence. Further research demonstrates that this mechanosensing strategy was applicable to different bacterial-cancer cell interactions. This study not only uncovered new cellular mechanotransduction mechanisms but also provided a versatile method that enabled in situ and dynamic indication of cancer cell responses to mechanical stimuli.

Despite recent advances in the understanding of relevance between microbes and cancer cells, the investigation of transformation from planktonic swimmer bacterial to sessile surface-attached bacterial remains a high priority.1-2 when bacteria encounters host cell surface, the first step is colonization and virulence induction.3 When it encounters lymphatic nonphagocytic tumor cells of T and B cell origin, it is recognized by phagocytic receptors and finally internalized. Bacteria can also been phagocyted by some kinds of cancer cells, such as breast cancer cells. Some investigators hypothesized that tumor cells acquire phagocytic properties during the course of malignancy.4 Nevertheless, the mechanism of this phenomenon was unclear and might be different from the common phagocytosis process. Besides the immune interactions, there are various interactions between mammalian cells and bacteria. It is proposed that the gut microbiome contributes to breast carcinogenesis.5-6 There are also some special interactions between non-phagocytic cells and bacteria, such as intestinal cancer cells and Escherichia coli (E.coli), which requires further investigation.7-8 Thus insight into the interactions between cancer cells and bacteria is of significant importance with respect to disease progression, spore detection for biodefense, and understanding cell clearance in general. In the case of the real physiological model, the contact between bacteria and cancer cells is accomplished under fluid shear stress (FSS). Studies have revealed that mechanical cues are perceived by bacteria during surface contact, leading to a rapid change in adherence, biofilm formation, surface motility, and virulence. 9-12 Meanwhile, internalization of the surface receptors of the cancer cells was also affected by FSS. The internalization of membrane proteins was mostly visualized by immunofluorescent labelling, which could only label one type of protein.13 A powerful method to observe and investigate the response of non-phagocyte phagocytosis dynamically under FSS is in great need. However, most methods for the detection of phagocytosis process bacteria inside macrophages rely on fluorescent labelling of the bacteria.14 15 These labels can be expensive, labile (requiring special storage and transport), and limited to a particular bacteria or target protein. In addition, commercial kits for labelling bacteria are also likely to label the cancer cells, thus they were usually used in abiotic conditions to avoid contamination.16 Furthermore, chemical modification of plasma membrane was more stable in FSS. The combination of DNA probes and microfluidic chips capable of generating FSS can be promising in biochemical

analysis.17 Moreover, in the phagocytosis process, a precise subcellular localization of biomolecules in plasma membrane would benefit the deeper insight into the recognition of bacteria. Labelling of biomolecules is a powerful tool for the investigation of sub-populations in the plasma membrane. 18-19 These approaches were mainly mostly based on chemical modification of reactive groups in the mammalian cell membrane,20 the biorthogonal reaction by metabolic labelling,21transfection of fluorescent protein, 22and hydrophobic group inserted into phospholipid bilayer.23-24 A wide range of biomolecules which might function in cancer cell membrane response to bacteria could be visualized in this way. Metabolic labelling could also tag surface immunomodulatory macromolecules of bacteria, which helps to track these inflammatory inducers of bacteria in the progression of intestinal diseases.25 But the behavior of membrane components of host cells during host-bacteria interaction under FSS still needs further research. Herein, we developed a DNA probe-based visualization system of biomolecules in the host cell membrane that allowed the observation of enhanced receptor recruitment on host cell membrane during host-bacteria interaction under FSS (Figure 1). To monitor the behavior and function of membrane proteins, cellsurface carbohydrate, and lipid bilayer during host cell-bacteria interaction, three different labeling strategy were utilized to respectively label the three components with fluorescent probes. Microfluidic chip was used for cancer cell culture and treatment, enabling precise flow control and real-time fluorescence observation.We found that when the cancer cells were exposed to shear stress generated by ﬂuid bacterial solutions, strong ﬂuorescence was emanated by the assembling of membrane proteins. Different responses of different probes indicated that membrane proteins, cell-surface carbohydrate, and lipid bilayer functioned in different manners. The proposed strategy was then applied to investigate the interactions among different types of bacteria and different types of host cells. All approaches described here on the model organism E.coli and Staphylococcus aureus (S. aureus) are amenable to application in the pathogens. These DNA probes thus offer a substantial technical advantage over traditional labelling methods for dynamic visualizing under FSS. EXPERIMENTS Materials. SU-8 2050 Negative photoresist and developer were purchased from Microchem Corporation (Newton, MA, USA).

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Figure 1. Enhanced receptor recruitment to bacteria under FSS in microchannels. (a) Bacteria interacted with cells under fluid shear stress in microfluidic channels. (b) Enhanced adherence of bacteria to cells revealed by strong fluorescence. (c) Phagocytosis of bacteria in static condition. PDMS prepolymers and initiators were purchased from Dow Corning (Midland, MI, USA). RPMI 1640 medium, trypsin, penicillin and streptomycin were obtained from Gibco Corporation (NY, USA). Sulfosuccinimidyl-6-[biotin-amido]hexanoate (NHSbiotin), N-azidoacetylmannosamine tetraacylated (ManNAz), lyso-tracker and mito-tracker were purchased from Thermo Scientific Co, Ltd (USA). Dibenzocyclooctyne-sulfo-Nhydroxysuccinimidyl ester (DBCO), Streptavidin (SA) was obtained from Millipore Sigma (Darmstadt, Germany). All DNA used in the experiment was synthesized by Sangon Biotech (Shanghai, China). Peptide was synthesized by Chinapeptides (Shanghai, China). Human breast adenocarcinoma cell line MCF-7 were purchased from Cancer Institute & Hospital Chinese Academy of Medical Science (Beijing, China). Dulbecoo’s Modified Eagle Medium (DMEM), (Gibco, 40 Grand Island, NY) and trypsin EDTA (Gibco, Grand Island) were purchased from Invitrogen (CA, USA). The E.coli solution was received from Beijing Forestry University. NH2 probe: 5’FAM-AAAAAAAAAA-NH2-3’ 5’Cy3AAAAAAAAAA -biotin-3’ Peptide probe: [K(N3)]-K-K(Pal)KK-K(Pal) (Remark: [K(N3)] indicates azide lysine, (Pal) indicates a hexadecanoic acid attached to the side chain of lysine. Cell culture on chip. MCF-7 cells were cultured in DMEM medium containing 10% FBS and 100 U/mL streptomycin and penicillin in a cell incubator with 5% CO2 at 37 °C. After the microchip was well-prepared, MCF-7 cells were first digested with trypsin, centrifuged and resuspended in new cell medium at a cell density of 106 cells/mL. The MCF-7 cell suspension was injected into the microchannel from the inlet and cultured for 45 min in the incubator. The inlets and outlets were covered by cell mediums to prevent evaporation, which would change the salt concentrations and affect cell viability. Cells were then cultured for 12 h and could be used for subsequent experiments. DNA labelling on cancer cell membrane proteins. MCF-7 cells in the chip were washed three times with phosphate buffer saline (PBS), and 2 mM NHS-biotin in PBS was injected into the microchannels and reacted at 4 °C for 30 min. MCF-7 cells were rinsed with PBS and incubated in streptavidin (50 μg/mL) at 4 °C for 20 min. After further washing, 2 μM 3'-biotin-labelled DNA probe (the probe was previously annealed, heated at 95 °C for 5 min, and slowly cooled to room temperature, named as NH2 probe)

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was added and reacted at 4 °C for 20 min. MCF-7 cells were then rinsed with PBS and refreshed with new cell medium. Synthesis of different kinds of probes. The DNA chain was obtained as the powder. Then the probe was resolved in PBS, with a final concentration of 10 mM. Then the DNA probe was mixed with DCBO-NHS (100 μM), incubating at room temperature for 30 min. After centrifuging in UF tube three times (10000 rpm), the mixture was purified and named as “click probe”. The peptide was solved in a mix solution (10 mM HEPES buffer, 300 mM mannitol and 20 μM DMSO). The peptide solution (70 μM in PBS) was added into the mixture, after which the reaction solution was incubated at 4 °C overnight. After centrifuging in UF tube three times (10000 rpm), the mixture was purified and named as “peptide probe”. Metabolic labelling of cells. MCF-7 cells cultured in the chip were first washed by PBS. Then new culture medium (1% FBS) with 50 μM ManNAz was added into the channels. The cells were incubated at 37 °C for 48 h. After washing with PBS three times, 2 μM click probe was added, reacted in 37 °C for 1 h. Cells were then rinsed with PBS and refreshed with new cell medium. Labelling of cells by hydrophobic effects. MCF-7 cells cultured in the chip were first washed by PBS. Then peptide probe (2 μM in PBS) was added into the channels, incubated at 37 °C for 10 min. Cells were then rinsed with PBS and refreshed with new cell medium. Bacteria culture. The E.coli strain and S. aureus was obtained in frozen stock solution. Then the strain was added into Luria-Bertani (LB) liquid culture medium. After incubating at 37 °C for 8 h, the medium was changed to DMEM culture medium with 10% FBS by centrifugation. The microbial suspensions were mixed in equal proportions, injected into microfluidic channels with a syringe pump for flow perfusion culture. RESULTS AND DISCUSSION Different types of probes labelling on MCF-7 cell membrane. During phagocytosis in immune cells, receptors in the host cell membrane first recognize pathogens, which cause membrane protrusions called pseudopodia to surround the target in a zipperlike mechanism26. To reveal the role of membrane proteins, cell-surface carbohydrate, lipid bilayer in bacteria-host cell interaction under FSS ， three kinds of DNA probes(NH2 probe, click probe, and peptide probe) were designed to label them. NH2 probe, click probe, and peptide probe was DNA sequence modified with biotin tag, azido group, and hexadecanoic acid, and they anchored to membrane proteins, cell-surface carbohydrate, and phospholipids ， respectively. The addition of DNA sequence could extend the labelling time of the probe on the host cell surface and attenuate endocytosis under FSS27-28. Their labelling procedure (Figure 2a) and labelling lifetime (Figure S1)were discussed later.

Figure 2. Labelling of three types of probes on the cell membrane. (a) Modification procedure of three different type of probes. (b) MCF-7 cells labelled with click probe. (c) MCF-7 cells labelled with peptide probe. (d) MCF-7 cells labelled with NH2 DNA probe Scale bar: 20 μm.


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Analytical Chemistry The NH2 probe was synthesized with a biotin tag, thus the NH2 probe could be annealed to streptavidin, which was chemically modified on host cell membrane proteins. This type of labelling was very stable with 4 h. Before the addition of click probe, the MCF-7 cells were incubated with ManNAz, which allows the cells bearing azido sialic acid residues in their plasma membrane. Then click probe could anchored to least 4 h due to the strong chemical bond between cyclooctyne group in DNA probe and azido group in MCF-7 cell membrane. The peptide probe insert into the phospholipid bilayer through hydrophobic interactions without additional processing (Figure. 2a). So this type of labelling vanished after 4 h due to high lipid fluidity of MCF-7 cell membrane. All these probes were sufficient to label the cell membrane and reveal the phagocytosis process(Figure. 2b,c,d) . Therefore, three different kinds of probes were used to indicate the response of different structures on the MCF-7 cell membrane to bacteria, respectively. FSS-enhanced membrane protein aggregation of MCF-7 induced by E.coli. It was proved that the MCF-7 cells could undergo apoptosis following phagocytosis of several strains of yeast.29 Nanoparticles could also be swallowed by MCF-7 cells. But the interaction between bacteria and MCF-7 cells was seldom discussed. Further investigation on this topic will be helpful for avoiding infections in cancer patients . The uniform labelling for membrane proteins by NH2 probe was first chosen for the test. In static condition (Figure 3a and 3b), MCF-7 cells cultured without E.coli showed a clear and uniform cell membrane profile. When MCF-7 cells cultured with E.coli, the intracellular bright spot indicated the accumulation of membrane protein within the MCF7cell. Bacteria also accumulated in large fluorescence spot and induced phagocytosis(Figure S2). This result was also been proved by E.coli stained by a commercial kit, SYTO® nucleic acid stains. It is a cell-permeant nucleic acid stain that show a large fluorescence enhancement upon binding nucleic acids. A free E.coli cell was caught by MCF-7 cell, and its fluorescence signal diminished with time passing by, compared with another free E.coli cell (Supplementary movie S1). The proposed DNA probe successfully meet the needs for investigation on plasma membrane protein during bacterial invasion process. In a biological environment, the bacteria lived in flow fluid before it transformed from planktonic swimmer microbe to sessile sur face-attached microbes1-2. Actually, bacterial dyes also stain MCF-7 cells in FSS, which is detrimental to observation (Supplementary movie S2). And the stains accumulated in MCF-7 cells over time, causing a strong fluorescence signal. Thus, it is impossible to reveal host cell membrane changes in real time, which limit their use in phagocytosis. Thus MCF-7 cells modified with NH2 probe was cultured in flow culture medium containing E.coli at the flow rate of 1 μL/min (FSS: 0.078 dyn/cm2). After 20 min, MCF-7 cells cultured without E.coli still remains a clear profile. When MCF-7 cells cultured with E.coli under FSS, plasma membrane protein accumulated into some cluster. (Figure 3c and 3d) This process can be viewed in the video in the Supplemental Information section (Supplementary movie S3, S4). Analyzed by Origin 9.1 software, an obvious fluorescence aggregation phenomena was observed (Figure 3e and 3f). Compared with Figure 3b, the irregular fluorescent region in MCF-7 cell membrane(Figure 3 d, f) indicated that the recruitment of membrane proteins induced by E.coli under fluid shear stress was significantly enhanced. The reason was estimated that the membrane receptors responded to the stimuli of E.coli under FSS. It was also reported that the FSS could help the residence time of microbes to cell surface27. The absence of intercellular red fluorescence indicated the phagocytosis process was interrupted and blocked at the receptor recognition stage. Previous publications

Figure 3. FSS-enhanced membrane protein aggregation of MCF-7 induced by E.coli. (a) MCF-7 cells labelled with NH2 probe. (b) MCF-7 cells labelled with NH2 probe co-cultured with E.coli under static condition. (c) MCF-7 cells labelled with NH2 probe under fluid shear stress. (d) MCF-7 cells labelled with NH2 probe cocultured with E.coli under fluid shear stress. (e) Distribution of fluorescence intensity of MCF-7 cells labelled with NH2 probe under fluid shear stress. (f) Distribution of fluorescence intensity of MCF-7 cells labelled with NH2 probe co-cultured with E.coli under fluid shear stress. Scale Bar, 10 μm.

confirmed that the fluid shear stress could strengthen the long-time binding of E.coli to monomannose-coated surfaces11. However, this is not exactly the same as the infective environment of bacteria in the organism. The hypothesis that main sensor of mechanical forces were type I fimbriae and motor proteins in microbes were also been widely accepted3, 12, 30. These studies ignored the changes in the host cells themselves when treated with the bacteria under FSS. The proposed probe was promising in real-time observation of host cell membrane protein recruitment process.And it also opens new avenues for dynamic revealing the extent of membrane protein aggregation. Response of cell-surface carbohydrate of MCF-7 cells infected by E.coli under FSS. Although breast cancer cells have been proved to have the phagocytic ability, the specific mechanism of phagocytosis has not yet been clarified. To further study this process, a metabolic labelling method was applied to specifically label cell-surface carbohydrate. Glycosyl labelling of MCF-7 cells was successfully achieved (Figure. 4a). Green fluorescence was evenly distributed on the MCF-7 cell membrane. In the experiment, the phagocytosis under static condition was also revealed by click probe. Unlike the NH2 probe, there was no obvious phagosome formation (Figure 4b). Actually, the phagocytosis was performed by polymerization and depolymerization of actin filaments, which sends pseudopods out to engulf the microbe and place it in a vesicle called a phagosome3132.The main role of glycosylated receptors in this process was to identify and anchor the bacteria so they would not accumulate in large amounts in the phagosomes. To investigate the role of cell-surface carbohydrate in the interactions between the bacteria and MCF-7 cell under flow condition, we first labelled the MCF-7 cells with the click probe and then continuously injected the culture medium containing E.coli into the microfluidic chip channel at a low flow rate (0.5 μL/min). With time passing by, the fluorescence of the MCF-7 cells


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Figure 4. Response of cell-surface carbohydrate and lipid bilayer of MCF-7 cells infected by E.coli under FSS. (a) MCF-7 cells labelled with click probe under static condition. (b) MCF-7 cells labelled with click probe co-cultured with E.coli under static condition. The white arrow indicated accumulation of cell-surface carbohydrate. (c) MCF-7 cells labelled with click probe under fluid shear stress. (d) MCF-7 cells labelled with click probe co-cultured with E.coli under fluid shear stress. Comparison of average fluorescence intensity (AFI) (e) and total fluorescence intensity (TFI) (f) of MCF-7 cells labelled with peptide probe co-cultured under FSS with E.coli, S. aureus, and their supernatant, respectively. Scale Bar, 10 μm.

cultured without E.coli gradually decreased, meanwhile, there were some clusters accumulated in the MCF-7 cell membrane. The clusters were also caused by the recruitment of receptor proteins (Figure 4c and 4d). Because most of the receptor proteins areglycoproteins. It is worth noting that the scattered green spots in Fig 4b are free E.coli. It is presumed that they interact with receptors on the MCF-7 cell membrane and seize the probe. The loss of fluorescence in MCF-7 cell membrane (Figure 4b, d) was due to the fluid shear stress would enhance the internalization of the membrane protein, the probes of the control group were gradually distributed in the membrane of the lysosomal and endosomal membrane. Thus the click probe could also reveal the recognition of pathogen. Response of lipid bilayer of MCF-7 cells infected by E.coli under FSS. With the aim of monitoring the lipid bilayer in phagocytosis, the peptide probe was employed to indicate the movement of the lipid bilayer. The DNA probe was conjugated with a peptide modified by two hexadecanoic acid chain, therefore they can easily anchor to the outer leaflet of the plasma membrane. The labelling result is shown in Figure 2c. The labeled MCF-7 cells were either treated with E.coli cell or E.coli culture supernatant under shear stress, and the cellular average fluorescence intensity (AFI) and total fluorescence intensity (TFI) were recorded along the time. The AFI of a cell referred to the intensity of fluorescence at each pixel and can indicate the degree of aggregation of the probe. TFI referred to the total amount of fluorescence of a cell, indicating the total amount of probes on the cell membrane. Under this labelling, the AFI of the MCF-7 cell surface barely changed when interacting with E.coli and E.coli supernatant (Figure 4e). This result indicated that there was no obvious

aggregation in lipid bilayer. Because lipid bilayer labelled with peptide probes do not have a signal recognition function like receptor proteins. But the TFI decreased significantly after the addition of E.coli cells (Figure 4f). Because this process took place very quickly and completed within 3 min after adding the bacteria to the start of the photograph. So the TFI of two groups at the first time point has been significantly different. But when we added the bacterial culture medium purified by the filter, there wasn’t obvious rapid weakening of fluorescence. So it was the microbe itself interact with MCF-7 cells rather than exotoxins. The main reason for this phenomenon was that a large amount of lipopolysaccharide was distributed on the outer membrane of E.coli, which helped to seize the peptide probe from MCF-7 cells. Different response from MCF-7 cells to E.coli and S. aureus under FSS. There are two main types of bacterial distinguished by the gram staining method. The difference in surface charge and flagellar between these two types of bacteria affects their interaction with the host cell under fluid. To study different interactions between mammalian cells with different types of microbes, E.coli and S. aureus were chose as representative of Gram-negative bacteria and Gram-positive bacteria32. MCF-7 cells were first labeled by peptide probes, and monitored in the presence of E.coli or S. aureus, under flow condition. Compared with E.coli, the S. aureus had a weaker interaction with MCF-7 cells, because it caused a lower fluorescence reduction effect than E.coli (Figure 4e and 4f). It is speculated that the fluorescence reduction effect was mainly due to the lipid exchange between MCF-7 cells and bacteria because later bacteria also emit green fluorescence33. Moreover, the degree of affinity of bacteria ／host cells can be explored by labelling cells with peptide probes. Peptide probes can indicate the affinity of different bacteria for host cells


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Analytical Chemistry

Figure 5. Different response from MCF-7 cells to E.coli and S. aureus under FSS. (a) MCF-7 cells labelled with NH2 probe treated with DMEM under fluidic shear stress. (b) MCF-7 cells labelled with NH2 probe treated with E.coli under fluidic shear stress. (c) MCF-7 cells labelled with NH2 probe treated under fluidic shear stress. Comparison of AFI(d), and TFI(e) of cells treated with S. aureus, E.coli and culture medium under static condition, revealed by NH2 probes. Scale Bar, 50 μm. due to weak binding. Because of the exotoxins secreted by S. aureus,MCF-7 cells gradually start to apoptosis, confirmed by Hoechst 33342 staining (Figure S3). While the interaction between E.coli and MCF-7 cells were thoroughly studied, it was still a puzzle for why S. aureus induced a weaker response in MCF-7 cells. Thus further investigation on S. aureus and MCF-7 cells was necessary. Therefore, the MCF-7 cells labelled with NH2 probes were treated with two types of bacteria, respectively. Under a flow rate of 0.5 μL/min, there were totally different responses of MCF-7 cells induced by two kinds of bacteria. The fluorescence intensity and area increased when MCF7 cells treated with E.coli, whereas the intensity decreased in the cell medium-treated control group. (Figure 5a and 5b) However, when the MCF-7 cells were interacted with S. aureus, the distribution area of the fluorescent probe decreased, but the AFI increased (Figure 5c-e). These results indicated that S. aureus could also induce an membrane protein-recruitment response, but the response was weaker than that of E.coli. The possible reason was that lipopolysaccharides (LPS) in E.coli was the main antigen recognized by mammalian immune cells, which was absent in S. aureus. In addition, the amount of lipoteichoic acid that can serve as an antigen is also low in S. aureus, thus it cannot cause a strong receptor recognition of MCF-7 cells.. What’s more, The peptidoglycan and staphylococcal protein A (SPA) in S. aureus can inhibit the phagocytosis. At the same time, S. aureus was more likely to take the probe off the MCF-7 cell surface, on account of the abundant teichoic acid present in the outer membrane, which binds to target MCF-7 cells non-specifically through membrane phospholipids. To explore this issue in more depth, click probe was employed to label the MCF-7 cell. Compared to MCF-7 cells treated with culture medium, MCF-7 cells treated with E.coli showed obvious membrane protein aggregation, and the cell outline vaguely remained. But there were no outlines of MCF-7 cells treated with S. aureus. Because most of the probes on the surface of MCF-7 cell

disappeared, the rest probes had a point-like distribution on the MCF-7 cell membrane (Fig S4). These results also illustrated that there was an interaction between S. aureus and MCF-7 cells, but the receptor-recruitment effect was weak. These results indicated that this series of the probe could successfully reveal the mechanism of S.aureus and E.coli respectively acting on MCF-7 cells. This method can help us not only observe the changes of MCF-7 cells in the phagocytosis in real time, but also observe the interaction between the two when the bacteria are not phagocytized by the host cells. Compared with the commercial membrane staining kit, it can precisely label different components of the plasma membrane, which play different roles in phagocytosis. Especially the NH2 probe was stable enough to avoid being seized by E.coli. This strategy is promising in the investigation on the receptor-recognition response of host cells, induced by different kinds of bacteria. CONCLUSION We successfully designed and constructed several DNA based probes anchored to different components of the plasma membrane by covalent and non-covalent interactions, providing approaches for in situ and dynamic monitoring of the interaction between microbes and mammalian cells. By this strategy, we found that fluid shear stress can enhance the adherence of bacteria to the MCF-7 cell surface, while inhibiting the phagocytosis of MCF-7 cells. The main mechanism was to enhance the recruitment of proteins in MCF-7 cell membranes. Moreover, the degree of aggregation of common membrane proteins and glycosylated membrane proteins was different, and MCF-7 cell membrane phospholipids have almost no aggregation during this process. DNA probes with different anchoring strengths indicate the strength of interaction between different bacteria and cells. Both of E.coli and S. aureus could induce the membrane protein aggregation under FSS, but to different degree. This work not only reveals the mechanosensing mechanism of action between tumor cells and bacteria with phagocytic ability but also provides a


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universal research method for studying inflammatory effects of cancer. For further investigation, the DNA probes can continue to be optimized to achieve the goal of simultaneously labelling multiple membrane components and specifically labelling on a certain protein to meet more precise research needs.

ASSOCIATED CONTENT Supporting Information Labelling of three types of probes on the cell membrane ； Attenuation of different kinds of probes ； Apoptosis of MCF-7 cells induced by S. aureus ；Different kinds of bacteria infected MCF-7 cells； Image acquisition and processing. (PDF) E.coli phagocytized by MCF-7 cells revealed by a commercial kit. (AVI) Supplementary Video S2: Interference from commercial kits in fluid shear stress. (AVI) Supplementary Video S3: MCF-7 cells labelled with NH2 probe co-cultured with E.coli under FSS. (AVI) Supplementary Video S4: MCF-7 cells labelled with NH2 probe cultured in culture medium under FSS. (AVI) The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax/Phone: +86 10 62792343. ORCID Wanling Zhang: 0000-0001-5154-3097 Jin-Ming Lin: 0000-0001-8891-0655

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21435002, 21727814 and 21621003) and National Key R&D Program of China (2017YFC0906800)

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In Situ Monitoring of Fluid Shear Stress Enhanced Adherence of

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