Instructed-Assembly as Context-Dependent Nanoscale Signals for Death and Morphogenesis of Cells
Context-dependent signaling, as a ubiquitous phenomenon in nature, is an inherent reactiondiffusion molecular process at nano-and microscales, but how to mimic its essence using noncovalent synthesis in cellular environment has yet to be developed. Here we show a dynamic continuum of non-covalent nanofibers formed by instructed-assembly (iA) of a supramolecular phosphoglycopeptide (sPGP) as context-dependent nanoscale signals for controlling death and morphogenesis of cells. Specifically, while enzymes (i.e., ectophosphatases) on cancer cells catalyze the formation of the nanofibers of the sPGP to result in cell death, damping the enzyme activity induces 3D cell spheroids. Similarly, relying on the ratio of stromal and cancer cells in a co-culture to modulate the expression of the ectophosphatase, the iA process enables cell spheroids. The spheroids act as a mimic of tumor microenvironment for drug screening. As the first demonstration of iA as multifunctional reaction-diffusion processes according to local enzyme activity for controlling cell behavior, this work illustrates context-dependent biological functions of non-covalent nanostructures in cellular environment.
Introduction<!>Results and discussion<!>Conclusion<!>Transmission electron microscopic<!>Cell culture<!>Cell viability assay<!>Live-dead assay<!>Time-dependent western blot
<p>This work reports the use of instructed-assembly 1 as context-dependent, active, and multifunctional nanoscale signals for controlling cell behavior. Being ubiquitous in nature, context-dependent signaling 2 plays major roles in many important cellular functions. For example, TNFR1, being activated by membrane-bound TNFα, induces cell death, but, interacting with secreted TNF-α, it leads prosurvival NF-κB signaling. 3 With advances on the molecular details of other TNF family ligands and receptors, now it is suggested that all of the death receptors exhibit such a context-dependent feature. 4 That is, the biochemical and biophysical nature of the interactions between these receptors and their extracellular ligands (or intracellular signaling partners) determines cell fate. In fact, many signaling pathways are context-dependent, such as TGF-β, 5 IKK-β, 6 Notch, 7 and Wnt-β-catenin 8 pathways, just to name a few. Despite the large diversity and daunting complexity of these context-dependent processes, they share several key common features: spatiotemporal regulated phosphorylation/dephosphorylation; 9 nanoscale higher-order organizations; 10 adaptive conformational transitions (or protein fuzziness). Particularly, the last two features have emerged as new insights in cell signaling processes. 10,11 In addition to advancing knowledge, these new insights of cell biology also provide guiding principles for developing synthetic molecules or materials that mimic the unique features of biological processes. However, how to use non-covalent nanostructures for mimicking the essence of context-dependent signaling remains less developed, 12,13 especially in live cells. [14][15][16][17][18] Our recent study on the dynamic continuum of supramolecular phosphoglycopeptides (sPGP) 19 implies that instructed-assembly (iA) likely would act as context-dependent nanoscale signals in cellular environment.</p><p>Instructed-assembly refers to forming ordered nanostructures of molecules as the consequence of a reaction, especially enzymatic reactions. 1 Like self-assembly, iA relies on non-covalent interactions to form large structures from small, simple building blocks; unlike self-assembly, iA includes a process away from equilibrium (G < 0), that is, the enzymatic reaction, and is inherently irreversible. In addition, the resulting nanostructures of iA highly depend on the path, that is, the history of molecular transformation. 1,20,21 More importantly, the intermediate status of instructed-assembly, which couples with reaction-diffusion processes 22 at nanoscales, plays key roles for determining the functions of resulted nanostructures. Although this concept appears obvious, it remains a challenge to use non-covalent nanostructures to form bioactive assemblies that exhibit multiple functions in a context-dependent manner.</p><p>Here we show a dynamic continuum 19,23 of non-covalent nanofibers formed by iA 1 of a sPGP as context-dependent signaling for controlling cell death and morphogenesis. As shown in Figure 1, while the enzyme (i.e., ALPL 24 ) on cancer cells (Saos-2 25 ) catalyze the formation of the nanofibers of the sPGP to result in cell death, damping the enzyme activity with a synthetic inhibitor of the enzyme induces 3D cell spheroids. Controlling the ratio of stromal (HS-5 26 ) and cancer (Saos-2) cells in a co-culture also modulates the expression of ALPL, which regulates the iA process to enable cell spheroids. Acting as a mimic of tumor microenvironment, the spheroids are useful for drug screening. Demonstrating iA as the multifaceted nanoscale signal according to local enzyme activity for controlling cell behavior, this work, for the first time, illustrates the promises of non-covalent nanostructures for context-dependent and active biological functions in cellular environment.</p><!><p>Using our recently developed sPGP (i.e., 1P:2, Figure 1A) as the building blocks, 19 we tested the response of Saos-2 cells to 1P:2. We chose Saos-2 cells and an uncompetitive inhibitor (DQB) 27 to inhibit the activity of the ALPL anchored on the plasma membrane of Saos-2 cell. Live-dead assay (Figure 2A and B) shows red fluorescence after 48 h incubation with 1P:2, indicating that most of the cells are dead. Co-incubating DQB (at a low concentration, 5 µM) with 1P:2 transformed the 2D cell sheet of Saos-2 to 3D cell spheroids, which exhibit green fluorescence, indicating that the cells are alive. Increasing the concentration of DQB results in a well-grown 2D cell sheet of Saos-2 (Figure S1). While incubating 1P:2 (500 M) induces about 50.0% death of Saos-2 cells, adding DQB (20 M) rescues the cells (about 90% viability). Either a pan-caspase inhibitor (zVAD-fmk) 28 or a PARP inhibitor (PJ34) 29 recuses cells from exposure to 1P:2 (Figure 2C), indicating that 1P:2 induces the apoptosis of Saos-2 cells. These results together suggest that iA, which generates the dynamic continuum of non-covalent nanostructures (containing 1P:2 and 1:2), controls cell death and morphogenesis depending on the activity of ALPL, that is, in a context-dependent manner.</p><p>To gain more insights into the transformation of 1P:2 to 1:2 on the cells that resulted in different cellular phenotypes, we investigated the enzymatic dephosphorylation rate of 1P:2 without or with the addition of DQB at different concentrations in the presence of Saos-2 cells. Liquid chromatography mass spectrometry (LC-MS) results show that 1P:2 undergoes ALPL catalyzed dephosphorylation at a much faster rate in absence of DQB (with t1/2 = 6.70 h), while the addition of 5, 10, and 20 µM DQB increases the t1/2 to 7.41, 9.37, and 10.9 h, respectively. After 24 h, the percentages of remaining 1P:2 are 3.3%, 16.9%, 26.5%, and 30.7% when the DQB concentrations are 0, 5, 10 and 20 µM, respectively. During this process, the activity of ALPL dictates the conversation of 1P:2 to 1:2, which further controls the cell behaviors. Moreover, co-incubating 1:2 with Saos-2 cells hardly changes the cell morphology and cell viability (Figure S4 and 5). These results suggest that the process of iA and the dynamic continuum of the nanostructures rather than the final structures determine the morphogenesis or death of the Saos-2 cells. Transmission electron microscopy (TEM) images show that 1P:2 exhibits different morphologies upon treatment of alkali phosphatase (ALP) at different concentrations: high concentration of ALP results in longer and uniform nanofibers with diameter of 8±2 nm (Figure 2E), while low concentration of ALP generates the mixture of nanofibers (8±2 nm) and nanoparticles (about 2 nm). These results imply that the morphology of the nanostructures formed by iA of 1P:2 shoud control the morphogenesis and fate of cells, which is useful for designing adaptive materials for controlling cell behaviors. To directly visualize the distribution of nanostructures formed by 1P:2, we used NBD-1P, a fluorescent analogue of 1P, 19 and 2 to co-incubate with Saos-2 cells. NBD is used because it acts as a sensitive fluorophore for reporting molecular self-assembly in cellular environment. 30 Being incubated with NBD-1P, Saos-2 cells only exhibit weak fluorescence on cell surface (Figure S6). The cells show bright fluorescence after being treated with NBD-1P:2 (Figure 3A), suggesting the non-covalent interactions between NBD-1P and 2 plays key role for generating the nanostructures on cell surface. Unlike the continuous fluorescent nanostructures on cell surface formed by NBD-1P:2 (Video clip S1), fluorescent dots appeared on cell surface when co-incubating NBD-1P:2 with DQB (Figure 3B and C, Video clip S2), indicating that the rate of dephosphorylation is a key control for iA to act as context-dependent nanoscale signals. This result agrees with the TEM image that 1P:2 forms nanoparticles and nanofibers at low concentration of ALP, while exhibits uniform nanofibers at higher concentration of ALP.</p><p>Recapitulating the complex features of cell processes in tissues, three dimensional (3D) cell models have served as a toxicity and activity assay of lead compounds in drug discovery for decades. 31 Among several osteosarcoma models, hanging drop cultures is the common strategy to induce 3D cancer cell spheroids for drug screening. But it is inefficient and requires long term culture for spheroid formation. 32 Being able to induce cell spheroids rapidly, this dynamic sPGP nanostructures should provide an alternative strategy for generating 3D cell models to assessing the efficacy of chemotherapy drug against osteosarcoma cells in vitro. 33 So we next evaluated the responses of Saos-2 cells, in 3D versus 2D, to different drugs. As shown in Figure 3, compared with 2D culture, Saos-2 cells in 3D culture show more resistance to anticancer drugs, such as doxorubicin (Dox), cisplatin, and taxol. Specifically, the IC50 of Dox against Saos-2 cells increases more than 3 times in 3D culture (> 6 µM, 48 h) than 2D culture (1.35 µM) after 48 h incubation. For cisplatin, the IC50 increases by 12 µM in 3D culture (33.25 µM, 48 h). For the Saos-2 cell treated by taxol (40 nM, 48 h), the cell viability increases from 60.8 % to 94.3% when the cells are cultured from as a 2D sheet to the 3D spheroids. These observations agree with the results obtained by using other 3D osteosarcoma models 33 and in vivo tumor models.  Although different strategies have been developed to produce tumor spheroids of a single cell type, it remains a challenge to mimic tumor microenvironment that consists more than one cell types. As a hallmark of cancer, heterogeneity is an essential property of tumor in vivo. Thus, it is important to develop a 3D co-culture of human tumor cells with different types of stromal cells and other cell types. 31 We next investigated whether iA of sPGP could induce spheroid formation in the co-culture consisting of normal stromal cells (HS-5) and cancer cells (Saos-2). Being treated with 1P:2 for 48 h, the 2D cell sheet of the co-cultured cells transforms to 3D cell spheroids, while the cells without any treatment or treated with one component (1P or 2) remain a 2D cell sheet (Figure S7). Co-incubation of DQB at higher concentration (10 µM, Figure 4B) abolishes the ability of 1P:2 to induce tumor spheroids, agreeing with that controlling activity of ALPL results in different cell phenotypes. Live-dead assay indicates that almost all the cells are live in the cell spheroid (Figure 4C and video clip S3). To visualize the distribution of normal cells and cancer cells within the 3D spheroids, we first labelled Saos-2 cells with a membrane dye 35 (green fluorescence) and HS-5 cells with nucleus dye (red fluorescence), and then co-cultured with 1P:2. As shown in Figure 4D (Video clip S4), Saos-2 cells and HS-5 cells interact with each other inside the cell spheroids. Western blot analysis (Figure 4E) indicates that the expression level of ALPL in co-culture system rises with increasing the percentage of Saos-2 cells in the co-culture of the cells. The ratio of HS-5 and Saos-2 cells in the co-culture controls the activity of ALPL, which further influences the process of iA of 1P:2 for the morphogenesis of the co-cultured cells. We also evaluated the differential drug response of the co-culture cells in 2D versus 3D. Both Dox and cisplatin exhibits dosage dependent cell toxicity in 2D cell sheets within the tested concentrations, while showing little toxicity in 3D cell spheroids. For example, 5 µM of Dox or 50 nM of cisplatin kills about half of cells in 2D cell sheets, but hardly causes cell death in the 3D cell spheroids.</p><!><p>In summary, this work describes that the iA of sPGP serves as a context-dependent nanoscale signal for controlling the death and morphogenesis of homotypic or heterotypic cells. Our work, for the first time, employs the process of non-covalent synthesis to mimic the signal transduction in a context-dependent manner, which advances the application of non-covalent nanostructures in cell milieu. 36 The processes of iA of sPGP at the cellular level, not only serve as a reactiondiffusion molecular process at nano-and microscales, 22 but also highlight the important role of iA for biological function. The use of iA for cell morphogenesis is significant because it provides a practical way to generate cell spheroids, which play increasingly important roles in biology and medicine. The concept demonstrated in this work may find applications in neural tissues or wounds, which use transient and context-dependent signals for functions. 37,38</p><!><p>After the grids (400 mesh copper grids coated with continuous thick carbon film ~35 nm in thickness) were glow discharged for 30 seconds, we placed sample solution on the grid (5 µL, sufficient to cover the grid surface). About 25 s later, we placed three large drops of ddH2O on parafilm to let the grid touch the water drops (each for 5 seconds) with the sample-loaded surface facing the parafilm, and then tilted the grid and gently absorbed water from the edge of the grid using a filter paper. Immediately after rinsing, we placed three large drops of uranyl acetate (UA) staining solution on parafilm to let the grid touch the staining solution drops with the sampleloaded surface facing the parafilm, and then tilted the grid and gently absorbed the stain solutions from the edge of the grid using a filter paper. After drying the grid, we examined the nanostructure on Morgagni 268 Microscope.</p><!><p>Saos-2 and HS-5 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Saos-2 cell lines were cultured in McCoy's 5A (Invitrogen Life Technologies) with 15% FBS and 1% P/S (100 U mL -1 penicillin and 100 μg mL -1 streptomycin, Invitrogen Life Technologies). HS-5 cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen Life Technologies) with 10% FBS and 1% P/S. Co-cultured cells of Saos-2 and HS-5 were cultured in McCoy's 5A with 15% FBS and 1% P/S. The incubation conditions for all the cells were at 37 °C in a humidified atmosphere of 5% CO2.</p><!><p>All cells were seeded in a 96-well plate with the density of 3×10 4 cells per-well (total medium volume of 100 µL). 24 hours post seeding, the solutions with a serial of concentrations (5 concentrations) of different precursors were added to each well. Cells without the treatment of the precursors were used as the control. At designated time (24/48/72 hours), 10 μL MTT solution (5 mg/mL) was added to each well and incubated at 37°C for another 4 h, and then 100 μL of SDS-HCl solution was added to stop the reduction reaction and dissolve the purple formazan. The absorbance of each well at 595 nm was measured by a multimode microplate reader. For 3D cell viability upon the treatment of different chemotherapy drugs, we first induced cell spheroid formation by 1P:2, and then replaced the culture medium with a serial of concentration of drugs. As comparing, 2D cell culture with the same cell number was used for parallel test. The cytotoxicity assay was performed three times, and the average value of the three measurements was taken.</p><!><p>Cell viability was performed by a live/dead assay. At designated times, the cells after the treatment were washed three times with PBS buffer, and then 100 μL of the Live/Dead solution containing 4 μM EthD-1 (ethidium homodimer-1) and 2 μM calcein AM was added on top of each sample for 30 min in an incubator at 37 °C with 5% CO2. After removing the staining solution, we used CLSM to view the sample with excitation filters of 450-490 nm (green, Calcein AM) and 510-560 nm (red, EthE). Green represented live cells, while red indicated dead cells.</p><!><p>After the co-cultured Saos-2 and HS-5 cells reached to about 75% confluence in 10 cm culture dish, cells were washed with PBS buffer for three times and then added 500 µL cell lysis buffer for 5 min. Cell scraper was used for harvesting all the lysis solution from the cell culture dish. The collected cell lysates were mixed with 5 μL of 100× proteinase inhibitor cocktail and then were snap-frozen and thawed for three cycles to lyse the cells. The cell lysates were then centrifuged at 12,000× g for 20 min at 4 °C, and the supernatant was collected. After quantification of proteins in cell lysates by the Bradford reagent, the same amount of proteins from the samples of different ratio of Saos-2 and HS-5 were mixed with the same volume of 2× Laemmli buffer before SDS-PAGE. After finishing the gel electrophoresis and transferring the proteins on the gels to the PVDF membranes for overnight, the membranes were incubated with primary antibodies (1:1000) for overnight. The membranes were then incubated with secondary antibodies (1:2000) for 1 h, followed by the reaction with ECL solutions for 10 min and detected by developing X-ray film.</p>