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Published in final edited form as: Nat Nanotechnol. 2023 May 22;18(9):1094–1104. doi: 10.1038/s41565-023-01401-7

Cell Spheroid Creation by Transcytotic Intercellular Gelation

Jiaqi Guo 1, Fengbin Wang 2,3,4,*, Yimeng Huang 1, Hongjian He 1, Weiyi Tan 1, Meihui Yi 1, Edward H Egelman 2,*, Bing Xu 1,*
PMCID: PMC10525029  NIHMSID: NIHMS1903281  PMID: 37217766

Abstract

Cell spheroids bridge the discontinuity between in vitro systems and in vivo animal models. However, inducing cell spheroids by nanomaterials remains an inefficient and poorly understood process. Here, we use cryo-EM to determine the atomic structure of helical nanofibers self-assembled from enzyme-responsive D-peptides and fluorescent imaging to show that transcytosis of D-peptide induces intercellular nanofibers/gels that potentially interact with fibronectin to enable cell spheroid formation. Specifically, D-phosphopeptides, being protease-resistant, undergo endocytosis and endosomal dephosphorylation to generate helical nanofibers. Upon secretion to the cell surface, these nanofibers form intercellular gels that act as artificial matrices and facilitate the fibrillogenesis of fibronectins to induce cell spheroids. No spheroid formation occurs without the endo- or exocytosis, the phosphate triggers, or the shape-switching of the peptide assemblies. This study, coupling transcytosis and morphological transformation of peptide assemblies, demonstrates a potential approach for regenerative medicine and tissue engineering.

Introduction

Cell spheroids (e.g., organoids1), bridging the discontinuity between in vitro systems and in vivo animal models,2, 3, 4 are better in vivo microenvironment5, 6, 7, 8 models than 2D layers of cells9 for mimicking human organs10, 11. Current procedures for generating spheroids,12, 13, 14, 15 such as non-stick substrates, are tedious, time-consuming, costly, or poorly controlled. None of these approaches mimics the characteristics of extracellular matrices (ECM) biogenesis, such as regulated precursor secretion and subsequent assembly into matrices16, to form intercellular junctions.17 Moreover, ECM proteins contain a variety of post-translational modifications (PTM) for enzymatic reactions to dynamically regulate cell adhesion18 and migration,19 such as high frequency of phosphorylation among the identified sites of ECM proteins.20 To date, it has been challenging for synthetic materials to mimic the dynamic and transient phosphorylation of ECM proteins. The inherently dynamic architecture of peptide assemblies21, 22, 23 formed by noncovalent interactions may provide a solution, as evidenced by that peptide assemblies form hydrogel matrices to mimic the ECM for cell culture24, 25 and tissue engineering.26, 27, 28 Most of the hydrogels, however, are made ex situ to enclose cells, which differ from tissues where cells surround the ECM made in situ. We recently have shown that enzyme-responsive D-phosphopeptide assemblies induce HS-5 cell spheroids29, 30, but the detailed mechanism remain obscure. Moreover, despite the diverse functions31 of peptide nanofibers, their atomic structures remain largely unknown.

Here, we report that transcytosis (i.e., endocytosis and exocytosis) of enzyme-responsive D-phosphopeptides creates intercellular D-peptide nanofibers as hydrogel matrices to enable cell spheroid formation (Figure 1a). The protease-resistant D-phosphopeptides undergo endocytosis and partial dephosphorylation during endosomal trafficking, which turn into nanofibers that serve as matrices of intercellular hydrogels upon exocytosis. Using cryo-EM, we determined the atomic structures of those D-peptide nanofibers responsible for intercellular gelation. The peptide nanofibers and fibronectin likely interact in secretory vesicles to enable the spheroid formation. Minimizing endocytosis, blocking the exocytosis, removing the phosphate triggers, or eliminating the nanoparticle-to-nanofiber transformation of the peptide assemblies abolishes the cell spheroid formation. Using protease-resistant yet enzyme-responsive peptides to mimic ECM biogenesis by generating helical nanofibers as intercellular gelation/matrices for ECM remodelling, this work illustrates a versatile approach that integrates transcytosis with enzymatic morphological transformation of peptide assemblies for potential applications in regenerative medicine and tissue engineering.

Fig. 1. Self-assembled D-peptide nanofibers form intercellular hydrogels.

Fig. 1.

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a, Schematic representation of the transcytotic dephosphorylation of D-peptide assemblies forming intercellular hydrogels that potentially interact with fibronectin and enable dispersed cells to form cell spheroids. b, NBD-ffspy contains three segments and its reaction in response to phosphatase. c, Optical images of NBD-ffspy (500 μM) in the in the absence or presence of ALP (0.1 U/mL) for 24 h in PBS. d, Time-dependent strain sweep of NBD-ffspy (500 μM) with ALP (0.1 U/mL) addition. e, Enzymatic conversion of NBD-ffspy (500 μM) in the presence of 1 U/mL ALP in PBS. f, TEM images of NBD-ffspy (500 μM) with ALP (1 U/mL) addition at designated time points.

Main text

Design of the peptide building block

Figure 1b shows the molecular structures of the D-phosphopeptide, which contains fluorescent nitrobenzoxadiazole (NBD) and a self-assembling D-phenylalanine-D-phenylalanine (ff).32, 33 NBD reports peptide assembly formation in cell assays, as reported previously.34 As serine and tyrosine are often enriched in the extracellular domain of integral membrane proteins, such as occludin for intercellular adhesion,35 we connected D-serine (s) and D-phosphotyrosine (py) at the C-terminus of ff. Phosphotyrosine endows the D-peptides with phosphatase-responsiveness. This design leads to the key molecule, NBD-ffspy (1). We also examined NBD-ffspy analogs with similar designs (Supplementary Figure 1a). According to the cryo-EM structures of the nanofibers of NBD-ffsy (vide infra), biphenyl-4-carboxylic acid (BP) or 2-naphthaleneacetic acid (Nap) replaces NBD to generate BP-ffspy (2) or Nap-ffspy (3), respectively. Since Claudin-1, another intercellular junction protein, has an increased extracellular proportion of serines and threonines,36 we doubled the number of D-serines or replaced D-serine with a D-threonine (t) for making Nap-ffsspy (4) and NBD-fftpy (5), respectively, to examine the role of serine or threonine bias for inducing spheroids. L-serine substitutes D-serine to form NBD-ffSpy (6) for examining the role of peptide configuration in inducing cell spheroids. Non-bulky D-alanine-D-alanine (aa) replaces ff to generate NBD-aaspy (7) for verifying the importance of ff motif. Switching the position of s and py generates a scrambled control, NBD-ffpys (8), for examining sequence specificity. NBD-ffsy (9) is for determining the importance of enzymatic dephosphorylation. We used solid phase peptide synthesis (SPPS) to produce these molecules (Supplementary Figure 1b) and confirmed their identities using LC-MS (Supplementary Figure 9-17).

Enzymatic gelation and self-assembly of NBD-ffspy

Treating NBD-ffspy (above its critical micelle concentration (CMC) (Supplementary Figure 2)) with alkaline phosphatase (ALP) leads to a hydrogel (Figure 1c). Strain sweep reveals ALP-instructed fast hydrogelation around 1.5 h, and confirms NBD-ffsy assemblies acting as gel matrices (Figure 1d and Supplementary Figure 3), agreeing with ALP rapidly converting NBD-ffspy to NBD-ffsy (Figure 1e). TEM images of NBD-ffspy at designated time points with ALP addition show that fibrils or other higher-order structures are absent before adding ALP. After introducing ALP, NBD-ffspy self-assembles into nanofibers and eventually form entangling networks after 24 h (Figure 1f). The bundle diameter gradually increases and becomes more dispersed over time (Supplementary Figure 4), agreeing that the NBD-ffsy assemblies are polymorphic (vide infra).

Cryo-EM structures of NBD-ffsy or BP-ffsy fibers

Cryo-EM imaging and reference-free 2D classifications of NBD-ffsy (formed by incubating NBD-ffspy with ALP for 24 h) confirms polymorphic cross-β filaments (Figure 2a) with four distinct species accounting for ~36%, 30%, 18% and 16% of total filaments (Supplementary Figure 5). Such polymorphisms are observed in almost all amyloid structures,37, 38, 39, 40, 41 such as KFE8.42 We tested all possible symmetries indexed from the averaged power spectrum (Figure 2c-d) by trial-and-error attempts until recognizable peptide-like features were found in each species. The most dominant filament species (class 1) has a helical rise of 0.48 Å and twist of 35.8°, with resolution of ~3.1 Å estimated by Fourier shell correlation (FSC) (Supplementary Figure 6). The filament reconstruction shows parallel cross-β packing of the NBD-ffsy molecules, with the heteroaromatic NBD pointing towards the center and the hydrophilic serine-tyrosine pointing outwards of the filaments (Figure 2f-g). The filaments are held together by extensive β-sheet hydrogen bonding and aromatic-aromatic stacking interactions between NBD and D-phenylalanine (Figure 2h-i). 3D reconstruction of the class 2 filaments reached ~3.2 Å resolution with a helical rise of 4.80 Å and a twist of −1.7°. The filament model possesses C3 symmetry, and each asymmetrical unit contains six copies of NBD-ffsy (Figure 2j-k). The cross-β packing of class 2 filaments depends on the hydrogen bonding between amides and π-π stacking among NBD motifs, displaying the carboxylic acid and hydroxyl group from tyrosine (Figure 2l-m). The polymorphic assemblies of NBD-ffsy include other two species with distinct molecular packings (Supplementary Figure 7). Although density appears at the C3-axis, the limited resolution of imaging prevents the unambiguous assignment of atomistic structures to the density maps of class 3 and 4 filaments.

Fig. 2. NBD-ffsy and BP-ffsy self-assemble into cross-β filaments in vitro.

Fig. 2.

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a, Cryo-EM image of four types of filaments formed by treating NBD-ffspy (500 μM) with ALP (1 U/mL) for 24 h. Green arrow: class 1; yellow arrow: class 2; blue arrow: class 3; red arrow: class 4. b, Cryo-EM image of filaments formed by treating BP-ffspy (500 μM) with ALP (0.1 U/mL) for 24 h. Average power spectra of c, NBD-ffsy class 1, d, NBD-ffsy class 2, and e, BP-ffsy filaments. f, 3D reconstruction, g, cross section and h, side view of NBD-ffsy class 1 filaments. Black arrow represents helical axis. i, Representation of an array of NBD-ffsy molecules along the axis in class 1 filaments. Dashed lines represent hydrogen bonding. j, 3D reconstruction, k, cross-section, and l, side view of NBD-ffsy class 2 filaments. m, Representation of an array of NBD-ffsy molecules along the axis in class 2 filaments. n, 3D reconstruction, o, cross-section, and p, side view of BP-ffsy filaments. q, Representation of an array of BP-ffsy molecules along the axis.

BP-ffsy, on the other hand, forms homogenous filaments that reach ~2.6 Å resolution (Supplementary Figure 8). Based on the averaged power spectrum (Figure 2e), 3D reconstruction of the filaments shows that the hydrophobic biphenyl motifs arrange at the center with C3 symmetry, a helical rise of 2.11 Å and twist of −54.8° (Figure 2n-o). Unlike NBD-ffsy, the tyrosines in BP-ffsy filaments point inward, and display C-terminal carboxylic acid and hydroxyl groups from serines (Figure 2p). BP-ffsy filaments also display cross-β packing and π-π stacking among biphenyl motifs (Figure 2q). Similarly, all three types of filaments display C-terminal carboxylic acid and hydroxyl groups on the surface, which may account for their spheroid-inducing property (vide infra).

Intercellular hydrogels for cell spheroid formation

HS-5 cells can serve as a reliable assay for examining the morphogenic effects of the D-peptide assemblies because they remains as single-layer individual cells during normal culture conditions, and hardly form cell clumps when overconfluent. At nontoxic concentrations (≤500 μM, Supplementary Figure 18), NBD-ffspy leads to spheroids in both adherent and suspended HS-5 cells (Figure 3a and 3b), with the extracellular distribution of assemblies within spheroids (indicated by white arrow). Notably, NBD-ffspy quickly induces aggregation in suspended cells within 12 h. The spheroid sizes correlate positively with the concentrations (Supplementary Figure 19a-b) and negatively with poly-D-lysine dish coating (Supplementary Figure 19c), suggesting that NBD-ffspy promotes cell-cell adhesion over cell-substrate adhesion. The spheroids gradually dissociate in fresh media, with the disappearance of intercellular fluorescence (Figure 3c) and fusion of some of the neighboring spheroids during dissociation (Supplementary Figure 20). The reversible cell aggregation agrees with the dynamic noncovalent interactions among the D-peptides, which “glue” the HS-5 cells together. Nuclei and live/dead cell staining confirms 3D cell aggregate formation (Figure 3d-e), accompanied by a necrotic core and a gradual decrease of viable cells from the periphery to the center of the spheroids (Figure 3f and Supplementary Figure 21). Hypoxia staining suggests a hypoxic core (Figure 3g-h). F-actin staining displays a cytoskeleton rearrangement with oriented distribution at the periphery, indicating spheroid compaction (Figure 3i). Cadherin staining reveals the cell-cell junctions, which are more evident in E-cadherin-abundant MCF-7 spheroids (Supplementary Figure 22). LC-MS analysis shows 20% NBD-ffspy and 80% NBD-ffsy in the assemblies (Supplementary Figure 23) from the spheroids. A mixture of them at this ratio forms nanofibers in PBS (Supplementary Figure 24).

Fig. 3. Spheroids from adherent and suspended HS-5 cells consist of intercellular hydrogel that colocalizes with fibronectin.

Fig. 3.

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a, DIC image and fluorescence 3D reconstruction of adherent HS-5 cells treated with NBD-ffspy (500 μM) for 24 h. b, Confocal images of suspended HS-5 cells treated with NBD-ffspy (300 μM) for 48 h. White arrow points to assemblies within spheroids. c, Phase contrast and confocal images of HS-5 cell spheroid disassembles in fresh medium within 72 h. d, Z-axis projection of nuclei and live/dead cell staining of the spheroids formed by treating suspended HS-5 cells with BP-ffspy (50 μM) for 72 h. e, 3D rendering of the live/dead cell staining of the spheroid in d. f, Spatial intensity profiles from the arrow indicated area in d. g, Hypoxia staining of HS-5 cell spheroids formed by treating with BP-ffspy (50 μM) for 72 h. h, Spatial intensity profiles from the arrow indicated area in g. i, F-actin staining of HS-5 spheroids formed by treating with BP-ffspy (50 μM) for 72 h. j, Representative confocal images of the FRAP assay on suspended HS-5 cells incubated with NBD-ffspy (200 μM) for 48 h. k, Quantitative analysis of the fluorescence intensity of the FRAP assay in j. l, Confocal images of the aggregation of suspended HS-5 cells in the presence of NBD-ffspy (500 μM) within 6 h. m, Zoom-in images of the NBD channel of HS-5 cells at designated time points. White arrows indicate pericellular assemblies. n, Immunofluorescence staining and o, corresponding colocalization scatter plots of adherent HS-5 cells treated with NBD-ffspy (500 μM) for 72 h and then fixed and stained with antibodies. p, Manders correlation coefficients of the fluorescence from NBD (green) and the fluorescence from the target protein (magenta). M1: fraction of green overlapping with magenta; M2: fraction of magenta overlapping with green. Data are presented as mean ± s.d. (n=3 independent measurements).

Fluorescence recovery after photobleaching (FRAP) shows that the intercellular fluorescence hardly recovers after photobleaching (Figure 3j), with a mobile fraction of 11.7% and a t1/2 of 4.94 s (Figure 3k), suggesting poor diffusivity. Co-incubating cells with NBD-ffspy and diffusive small-molecule probes (rhodamine B or Nile red) displays trapped probes with poor recovery (Supplementary Figure 25 and 26), indicating intercellular hydrogelation. DIC images show scattered cells at 0 h and cell aggregates after 6 h (Figure 3l). The individual cells show random motion within 0-3 h and become “sticky” and undergo gradual aggregation from 3-6 h (Supplementary Video 1). NBD channel displays peri/inter-cellular D-peptide assemblies (indicated by white arrows) from 2-6 h, which occurs simultaneously with morphogenesis (Figure 3m). Immunofluorescence staining of various ECM proteins, surface receptors, and cytoskeleton on NBD-ffspy treated HS-5 cells shows that the peptide assemblies colocalize with several ECM proteins (Figure 3n-o and Supplementary Figure 27), especially with fibronectin that has sufficient signal intensities to achieve good colocalization for both M1 (fraction of NBD overlapping with protein) and M2 (fraction of protein overlapping with NBD) (Figure 3p). Collectively, these results indicate that NBD-ffspy undergoes dephosphorylation to form intercellular hydrogels that colocalize with fibronectin to promote cell-cell adhesion and generate spheroids.

BP-ffspy allows enzymatic dephosphorylation while enhances self-assembling ability to form nanofibers at low concentrations. BP-ffspy induces large spheroids with a tenfold decrease of its effective concentrations (25-100 μM) (Supplementary Figure 28a-d). The spheroids induced by BP-ffspy traps small-molecule probes (Supplementary Figure 28e-f), suggesting in situ hydrogelation. Nap-ffspy, Nap-ffsspy, and NBD-fftpy display similar morphologies of nanofibers after dephosphorylation (Supplementary Figure 29c) and induce HS-5 cell spheroid formation (Supplementary Figure 29a-b). NBD-ffpys only induces small spheroids in suspended cells at high concentrations, which correlates with NBD-ffys being a weak hydrogelator (Supplementary Figure 29d). NBD-aaspy or NBD-ffSpy abolishes the nanoparticle-to-nanofiber morphological transformation upon ALP addition. Neither of them induces cell spheroids (Supplementary Figure 29a-b). Pre-assembled NBD-ffsy nanofibers, forming chunks of hydrogels deposited on cell surface, fail to induce spheroids (Supplementary Figure 29e and 30). Mixing NBD-ffspy with NBD-ffsy generates pre-assembled fibrils and hydrogel chunks that undermines spheroid-inducing ability (Supplementary Figure 31). These results suggest the crucial role of morphological transformation and in situ hydrogelation for cell spheroid formation.

Transcytosis of D-peptide assemblies

Inspired by the synthesis of cellular fibronectin that involves secretion of soluble protein dimers and subsequent self-assembly into insoluble matrix,43 we examined the roles of transcytosis in spheroid formation. We first used endocytosis inhibitors of distinct pathways and found that chlorpromazine, a clathrin-mediated endocytosis inhibitor, significantly reduces cellular uptake of NBD-ffspy (Supplementary Figure 32). Due to toxicity of CPZ to HS-5 cells (Supplementary Figure 33a), we turned to tamoxifen-induced dynamin tripe knockout (TKO) mouse fibroblast to reduce endocytosis. After verification of successful knockout (~90%) (Supplementary Figure 34a-b), confocal images show reduced number of vesicles per cell accompanied with decreased sizes of spheroids, suggesting the crucial role of endocytosis in spheroid formation (Figure 4a-d and Supplementary Figure 34c).

Fig. 4. Endocytosis and exocytosis are crucial for spheroid formation.

Fig. 4

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a, Confocal images of TKO cells with or without tamoxifen treatment incubated with NBD-ffspy (200 μM) for 1 h. b, The number of vesicles per cell in a. Data are presented as mean ± s.d. (n=20 cells/group). c, Suspended TKO cells with or without tamoxifen incubated with NBD-ffspy (200 μM) for 48 h. d, The diameters of the TKO spheroids with or without tamoxifen treatment induced by NBD-ffspy (200–500 μM). Data are presented as mean ± s.d. (n=10 spheroids/group). e, Confocal images of adherent HS-5 cells with NBD-ffspy (500 μM) under different treatments for 48 h: Control (NBD-ffspy); + Ver (NBD-ffspy + 20 μM Ver); + BFA (NBD-ffspy + 50 nM BFA); + V-1 (NBD-ffspy + 10 μM V-1); Sucrose (NBD-ffspy). f, Confocal images of suspended HS-5 cells under different treatments for 48 h (NBD-ffspy at 200 μM due to removed cell-substrate adhesion): Control (NBD-ffspy); + Ver (NBD-ffspy + 50 μM Ver); + BFA (NBD-ffspy + 300 nM BFA); + V-1 (NBD-ffspy + 10 μM V-1); Sucrose (NBD-ffspy). g, Confocal images of LAMP-1-RFP labelled HS-5 cells incubated under the same condition as cells in e. Sucrose pre-treated cells: HS-5 cells preincubated with 0.1 M sucrose for 72 h. h, Manders correlation coefficients of the green and magenta fluorescence from cells in g. M1: fraction of green overlapping with magenta. Data are presented as mean ± s.d. (n=3 independent measurements).

We used three types of exocytosis inhibitors to inhibit secretion: (i) verapamil (Ver) for inhibiting calcium-dependent exocytosis; (ii) brefeldin A (BFA) for inhibiting conventional endosomal trafficking; (iii) vacuolin-1 (V-1) and sucrose for inducing vacuolation, with V-1 preventing unconventional endo-lysosomal secretion44. Treating HS-5 cells at nontoxic concentrations (Supplementary Figure 33b-d), all the three inhibitors undermine spheroid formation in both adherent and suspended HS-5 cells (Figure 4e-f). Moreover, the exocytosis inhibitors abolish spheroid formation in a previously reported spheroid-inducing D-peptide, Nap-ffk(biotin)py (Supplementary Figure 35), demonstrating the generality of this exocytotic mechanism in generating spheroid. Unlike the uninhibited cells that display extracellular assemblies, inhibitor treated groups exhibit reversible intracellular distribution of NBD-ffspy puncta, suggesting endosomal localization of the D-peptides (Supplementary Figure 36). NBD co-localizes with LAMP-1-RFP after Ver, BFA, or sucrose treatment, while uninhibited cells display less colocalization, further supporting the NBD-ffspy endosomal localization. V-1 significantly induces vacuolation and prevents endosome-lysosome fusion, which results in poor colocalization as well (Figure 4g-h and Supplementary Figure 37). These results support that NBD-ffspy induce spheroids by transcytosis of the D-peptide filaments to the intercellular space.

To determine whether the dephosphorylation happens extracellularly by ecto-phosphatases or intracellularly during endosomal trafficking, we added ALP to culture media. Extracellular ALP addition significantly reduces intercellular assemblies and cell aggregation, unless diluting ALP to ultra-low levels (e.g., 0.001 U/mL) (Figure 5a and Supplementary S38). Moreover, inhibiting tissue-nonspecific ALP (TNAP) overexpressed by SJSA-1 cells significantly increases the sizes of spheroids for SJSA-1 treated by NBD-ffspy or BP-ffspy (Figure 5b-c). TNAP inhibition results in colocalization of peptide fibers with fibronectin, but the fibers form throughout the cells and poorly localize with fibronectin in uninhibited SJSA-1 (Figure 5d-e). These results support that NBD-ffspy mainly undergoes intracellular dephosphorylation during endosomal trafficking to generate intercellular assemblies and indicate that extracellular dephosphorylation disfavor fibronectin colocalization to hamper spheroid formation.

Fig. 5. Intracellular dephosphorylation is crucial for spheroid formation.

Fig. 5

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a, Confocal images of adherent HS-5 cells treated with NBD-ffspy (400 μM) in serum-free DMEM for 24 h with different concentrations of ALP addition. b, Phase contrast images of suspended SJSA-1 cells incubated with NBD-ffspy (400 μM) or BP-ffspy (100 μM) in the presence or absence of TNAP inhibitor (5 μM) for 48 h. c, The diameters of SJSA-1 spheroids in b. Data are presented as mean ± s.d. (n=10 spheroids/group). d, Immunofluorescence staining of adherent SJSA-1 cells treated with NBD-ffspy (500 μM) in the presence or absence of TNAP inhibitor (5 μM) for 72 h. White arrows indicate fibronectin. e, Manders correlation coefficients of the green and magenta fluorescence from cells in d. M1: fraction of green overlapping with magenta. Data are presented as mean ± s.d. (n=3 independent measurements). f, Quantification of the sizes of the spheroids formed by treating suspended cells with NBD-ffspy (500 μM) for 48 h. Data are presented as mean ± s.d. (n=20 spheroids/group). g, Phase contrast images of suspended U-87 MG cells in the treatment of NBD-ffspy (300–500 μM) for 24 or 48 h. h, Z-axis projections of immunofluorescence staining of fibronectin in various cell lines.

We incubated NBD-ffspy with nine additional cell lines expressing different levels of phosphatases for evaluating the generality. Enzymatic dephosphorylation of NBD-ffspy by cells displays fast conversion in Saos-2, SJSA-1 and HeLa, accompanied with membrane deposition of assemblies and decreased cell viability (Supplementary Figure 39), similar to the previous results45, 46. Spheroid sizes differ among the cell lines. HS-5 and U87MG cells form large spheroids while PC-3, MCF-7, OVSAHO, SKOV-3, HepG-2, and SJSA-1 cells form smaller ones. HeLa and Saos-2 cells hardly form cell aggregates. U-87 MG spheroids grow over time, like HS-5, in a concentration-dependent manner. (Figure 5f-g and Supplementary Figures 39b-c, 40a).

Immunostaining shows cell type-specific morphologies of fibronectin, with HS-5, SJSA-1, and U-87 MG mainly display fibrillar fibronectin, while SKOV-3, MCF-7, and HepG-2 show globular fibronectin. HeLa, OVSAHO, Saos-2, and PC-3 display weak signal. These results correspond to the tissue-dependent isoforms of fibronectin, such as soluble plasma fibronectin by hepatocytes and insoluble cellular fibronectin by fibroblasts.43 NBD-ffspy is more potent among cells expressing fibrillar fibronectin and low phosphatase levels (Figure 5h and Supplementary Figures 40b-d). BP-ffspy induces spheroids of SJSA-1 and SKOV-3, with the formation of necrotic core (Supplementary Figures 41). We also co-incubated HS-5 with other cell lines to introduce fibrillar fibronectin and mimic the tumor microenvironment. BP-ffspy induces heterotypic spheroids in HS-5 with PC-3-DsRed, Saos-2-GFP, SJSA-1-RFP, or HeLa-GFP, with the fluorescent cancer cells distributing throughout the spheroids (Supplementary Figures 42). These results support the crucial role of fibrillar fibronectin in spheroid formation.

D-peptide nanofibers facilitate fibronectin fibrillogenesis

We conducted live cell imaging of HS-5 cells incubated with rhodamine-fibronectin (rFN) and NBD-ffspy. HS-5 quickly process the rFN derived from plasma to form fibrillar structures, while NBD-ffspy undergoes punctum-to-fibril transformation and gradual colocalization with rFN (Figure 6a). The sizes of rFN increase as they colocalize with NBD-ffspy in 24-72 h, indicating NBD-ffspy-facilitated fibronectin remodeling. NBD-ffspy and rFN exhibit the early partial overlap in endosomes in 4 h, followed by the initiation of intercellular colocalization in 24 h, which gradually achieved good overlay in 48-72 h (Figure 6b-d). At cell free condition, incubating rFN with ALP or NBD-ffspy retains the globular structure, while co-incubation with nanofibers of NBD-ffsy, either pre-assembled (NBD-ffsy) or enzymatically formed (NBD-ffspy + ALP), facilitates the fibrillogenesis of rFN (Figure 6e). NBD-ffspy-induced crowding likely promotes rFN aggregates, which transform to radiating nanofibers from aggregates upon ALP addition. Reducing initial NBD-ffspy concentrations decreases the sizes of aggregates (Supplementary Figure 43). Colocalization analysis reveals rFN with background fluorescence from NBD-ffspy in the absence of ALP (Figure 6f), while incubating with NBD nanofibers increases colocalization (Figure 6g-h). The enzymatic nanofibers are invisible under confocal microscopy, suggesting finite and homogeneous nanostructures. Therefore, we pre-treated NBD-ffspy with ALP to generate enzymatic nanofibers, and then incubated with rFN. Confocal images show colocalization with evident fibrillogenesis of rFN, supporting the ECM remodeling effects of NBD-ffsy nanofibers (Figure 6i-k). The more potent spheroid-inducing analog, BP-ffspy, when pre-treated with ALP to form enzymatic nanofibers, induces curly fibers of rFN that entangle into 3D networks (Figure 6l-m). The fibrillogenic concentrations correspond to its spheroid-inducing concentrations, where high concentrations of BP-ffspy lead to large chunks of rFN fibrils and fail to induce spheroids (Figure 6n and Supplementary Figure 28d).

Fig. 6. D-peptide nanofibers facilitate the fibrillogenesis of fibronectin.

Fig. 6

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a, Confocal images of adherent HS-5 cells treated with rhodamine-fibronectin (rFN) (5 μg/mL) and NBD-ffspy at various time points. Spatial intensity profiles of HS-5 cells in the magnified area in a incubated for b, 4 h; or c, 24 h. d, Colocalization scatter plots of the fluorescence intensities from cells in a incubated for 48 h or 72 h. e, Confocal images of samples incubated at 37 °C for 24 h: rFN + ALP (upper left); NBD-ffsy (middle left); NBD-ffspy + ALP (lower left); rFN + NBD-ffspy (upper right); rFN + NBD-ffsy (middle right); rFN + NBD-ffspy + ALP (lower right). Spatial intensity profiles of f, rFN + NBD-ffspy; g, rFN + NBD-ffsy or h, rFN + NBD-ffspy + ALP in e. i, 3D rendering of NBD-ffspy treated with ALP for 48 h and then incubated with rFN for 9 h. j, Z-axis projection of the stack in i. k, Colocalization scatter plot of fluorescence intensities form two channels in j. l, 3D rendering of BP-ffspy treated with ALP for 48 h and then incubated with rFN for 24 h. m, One slice of confocal image from l. n, Z-axis projection of BP-ffspy (500 μM) treated with ALP for 48 h and then incubated with rFN for 24 h. o, TEM images of rFN incubated with NBD-ffspy (100 or 400 μM) in the presence of ALP for 48 h. Red arrows indicate nanofibers with increased diameters. p, TEM images of BP-ffspy treated with ALP for 48 h and then incubated with fibronectin (50, 25 or 5 μg/mL). [rFN] =50 μg/mL, [NBD-ffsy] =500 μM, [NBD-ffspy] =500 μM, [BP-ffspy] = 50 μM, [ALP] =0.1 U/mL unless indicated otherwise.

We used TEM to image NBD-ffspy with rFN in the presence of ALP. While 100 μM of NBD-ffspy results in amorphous aggregates, wider nanofibers (indicated by red arrows) with helical features (Figure 6o) appear at 400 μM of NBD-ffspy. Starting from 50 μM of BP-ffspy treated with ALP, the TEM images show close-packed bundles of nanofibers with 50 or 25 μg/mL of fibronectin. Decreasing to 5 μg/mL of fibronectin leads to loose-packed nanofibers with aggregates in between (Figure 6p). The fibronectin-dependent morphology indicates the potential interactions of the peptide assemblies with fibronectin.

Conclusion

In brief, we examined the structures and mechanism of D-peptide nanofibers formed at the intercellular space of HS-5 cells, which form spheroids quickly in both adherent and suspended conditions. Our findings showed that the self-assembling motif, phosphate, and nanoparticle-to-nanofiber transformation are indispensable for spheroid formation. Having a phosphotyrosine residue, NBD-ffspy undergoes transcytotic dephosphorylation to create intercellular assemblies that colocalize with fibronectin. These peptide assemblies facilitate the fibrillogenesis of fibronectin and act as reversible intercellular nanojunctions that “glue” cells together. By forming an adaptive supramolecular hydrogel47 that remodels endogenous proteins and controls reaction-diffusion at the nano- and microscale,48, 49 this work illustrates the potential of integrating cryo-EM, enzymatic shapeshifting (nanoparticle-to-nanofibers), and cell biology (endocytosis and exocytosis) to mimic ECM biogenesis. Based on the phenotype switch in the differentiation of stem cells,50 the approach taken in this work should be useful for guiding cell differentiation, which is a subject of ongoing study.

Methods

Materials and instruments

Details of this section are provided in the Supplementary Information.

Peptide synthesis (NBD-ffspy as an example)

All the peptides were synthesized according to standard SPPS procedures using 2-chlorotrityl resin. Fmoc-O-phospho-D-tyrosine and NBD-β-alanine were synthesized according to reference.51, 52 The peptides were cleaved using a cleavage cocktail (95% TFA, 2.5% triisopropyl silane, and 2.5% H2O) for 1 h. After concentration, ice-cold ethyl ether was used for peptide precipitation. The crude peptides were purified by HPLC.

Determination of critical micelle concentration (CMC)

The CMCs were determined using pyrene as an environment-sensitive probe.53 Samples of a series of concentrations (3.1 μM-2.0 mM) were prepared in pyrene-saturated PBS. The fluorescence emission spectrum of each concentration was collected. The peak intensities of 373 nm (I1) and 383 nm (I3) were obtained to calculate the I1/I3 value. I1/I3 values were plotted against concentrations of samples. CMC was determined as the intersection of two linear regression curves.

Rheology test

Time-dependent strain sweeps were conducted at room temperature on 400 μL of NBD-ffspy (500 μM) with ALP (0.1 U/mL) in PBS at the frequency of 6.28 rad/s and the strain of 0.1%, with the scanning frequency of 10 min/point. Frequency and strain sweeps were then conducted on the hydrogel formed. Frequency sweeps were conducted at the strain of 1%. Strain sweeps were conducted at the frequency of 6.28 rad/s.

Transmission electron microscopy

400-mesh copper grids coated with carbon film were glow discharged at −20 mA for 30 s. 3 μL of sample was loaded onto a grid and let stand for 1 min. Use filter paper to remove any excess of sample. The sample was stained with 2% uranyl acetate for 20 s, then imaged on Morgagni 268 transmission electron microscope at a high voltage of 80 kV.

Cryo-EM microscope and image processing

The peptide nanofiber sample was applied to glow-discharged lacey carbon grids and vitrified using a Leica plunge freezer (Leica) or a Vitrobot (Thermo Fisher). Grids were imaged on a Titan Krios (300 keV, Thermo Fisher) with a K3 camera (Gatan). Micrographs were collected under electron counting mode, using a defocus range of 1-2 μm with ~50 electrons/Å2 distributed into 40 fractions. Motion correction and CTF estimation were done in cryoSPARC54, 55, 56. Particles were auto-picked by “Filament Tracer” with a shift of ~9 pixels. Non-peptide junk particles were removed by multiple rounds of reference-free 2D classifications. Particles were kept if they had clear 2D average patterns. Based on their shape and diameters, NBD-ffspy were then grouped into four different classes (class 1, class 2, class 3, and class 4). BP-ffsy, on the other hand, form homogenous nanofibers and only one type of fibers was observed. For each class of NBD-ffspy, the possible helical symmetries were calculated from an averaged power spectrum of the raw particles. All possible symmetries were then tested by trial and error in cryoSPARC until recognized peptide features, such as density of side chains, were observed. The resolution of each reconstruction was estimated by both Map:Map FSC and Model:Map FSC. The final volumes were then sharpened with a negative B-factor automatically estimated in cryoSPARC, and the statistics are listed in Supplementary Table 1 and 2.

Model building of NBD-ffsy filaments

The class 1 filament reached the highest resolution at ~3.1 Å among all the four different classes. The class 2 filament reached a resolution at ~3.2 Å based on map:map FSC. Since the NBD-ffsy filaments are made of only β-sheets, the hand of the helical map cannot be determined directly from the cryo-EM volume. This is unlike volumes that contain an α-helix, in which the hand is obvious when the resolution is 4.5 Å or better. In the published cross-β structures, the parallel β-sheets typically have a left-handed twist. However, this observation may not be deducible to short peptide containing non-standard residues. Therefore, we did model building in both hands of the map of class 1 filament. First the model was manually adjusting in Coot57 and then real-space refined in PHENIX58. It turned out the model fits in left-handed 10-start map slightly better than right-handed 10-start map, with RSCC 0.89 vs 0.85 and Clashscore 22 vs 80. Therefore, we suggested the map of class 1 filament likely has a left-handed 10-start. Similar procedures were performed for class 2 filament. The class 3 and 4 filaments reach a moderate overall resolution of ~3.6 Å, but the resolution is not enough to determine the hand of the map or build unambiguous atomic models. The resolutions for all four classes and their refinement statistics are shown in Supplementary Table 1.

Model building of BP-ffsy filament

The hand of the BP-ffsy filaments was determined in a similar approach as described above. According to map:map FSC, 3D reconstructions of BP-ffsy reach 2.6 Å resolution. To refine the model into the cryo-EM map, we first made geometry restraints of the compound eLBOW. Then, the model was manually adjusted in Coot57 and real-space refined in PHENIX58. The refinement statistics of both filaments are shown in Supplementary Table 2.

Cell culture

HS-5, HeLa, SJSA-1, SJSA-1-RFP, Saos-2, SKOV-3, PC-3, MCF-7, HepG-2, and U-87 MG cells were purchased from ATCC, OVSAHO from Cellbank (Japan). TKO cells were given by Prof. Pietro De Camilli group. HeLa-GFP, Saos-2-GFP, and PC-3-DsRed were given by Dr. J. T. Hsien. HS-5, HeLa, Saos-2, and HepG-2 cell lines were authenticated by CellCheck 9 - human (9 Marker STR Profile and Inter-species Contamination Test, IDEXX), confirming 100% match of the cell identity. HS-5 and TKO cells were cultured in DMEM supplemented with 10% FBS. HeLa, HeLa-GFP and HepG-2 cells were cultured in MEM supplemented with 10% FBS. SJSA-1, SJSA-1-RFP and OVSAHO cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Saos-2 and Saos-2-GFP cells were cultured in McCoy’s 5A medium supplemented with 15% FBS. SKOV-3 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS. U-87 MG cells were cultured in EMEM supplemented with 10% FBS. MCF-7 cells were cultured in EMEM supplemented with 10% FBS and 0.01 mg/mL human recombinant insulin. PC-3 and PC-3-DsRed cells were cultured in F-12K supplemented with 10% FBS. All the cell lines were supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin and were cultured and humidified with 5% CO2 at 37 °C.

Confocal microscopy of the spheroids

Adherent cells

Cells were seeded at 1.5×105 cells per dish on the inner 2 cm glass of a 3.5 cm confocal dish for 24 h to allow attachment. The peptide-containing fresh culture medium (500 μL) at working concentration was prepared directly from the stock solution (10 mM) in PBS. After incubating cells with culture medium for a designated period of time, live cell imaging buffer was added to replace the culture medium. The cells were then used for live cell imaging.

Suspended cells

Cells were treated with trypsin to obtain a cell suspension at a concentration of 1.5×105 cells per milliliter in culture medium. Peptide stock solution was added into the culture medium to obtain a working solution with suspended cells. Cells were seeded at 7.5×104 cells per dish on the inner 2 cm glass of a 3.5 cm confocal dish for incubation. After incubating cells for a designated period of time, live cell imaging buffer was added to replace the culture medium. The cells were then used for live cell imaging.

Poly-D-lysine dish coating

In the biosafety cabinet, coat confocal dishes with poly-D-lysine at desired concentrations (0.050-50 μg/mL) by direct dilution from the stock solution (1 mg/mL) using sterile water. Incubate the confocal dishes at 37 °C for 1 h and aspirate the solution. Rinse with sterile water and allow the surface to dry before cell seeding.

Hypoxia and Live/dead staining of spheroids

HS-5 cells were seeded at a density of 7.5×104 cells per dish in the treatment of BP-ffspy (50 μM) and incubated for 72 h under normoxic conditions. The spheroids were the stained with 5 μM hypoxia probe and 1 μg/mL Hoechst 33342 for 1 h for hypoxia staining, or 2 μM calcein AM and 4 μM ethidium homodimer-1 for 1 h for live/dead imaging, respectively.

Immunofluorescence staining

Remove the culture media from cells and wash twice with PBS. The cells were fixed with warm 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Image-iT™ FX signal enhancer was added and incubated for 30 min to eliminate nonspecific binding with the dye molecules. The cells were then blocked with 3% BSA in PBS for 1 h at room temperature. Cells were stained with primary antibodies at distinct dilution in 1% BSA in PBS overnight at 4 °C, and then incubated with Alexa Fluor conjugated secondary antibody at a dilution of 1:1000 for 1 h at room temperature. Nuclei were stained with 1 μg/mL of Hoechst 33342 in PBS for 15 min. Wash with PBS three times after each step, except after blocking, where primary antibodies were directly added.

Dilution for the primary antibodies: Laminin, 1:100; fibronectin, 1:200; E-cadherin, 1:50; N-cadherin, 1:100; integrin alpha 5, 1:250; integrin beta 1, 1:200; integrin beta 3, 1:200; collagen I, 1:100; collagen III, 1:100; collagen IV, 1:100; EGFR, 1:200, TGF beta 1, 1:200; Phalloidin-Alexa Flour 633, 5 U/mL (without the incubation of secondary antibody).

Determination of dephosphorylation by ALP

NBD-ffspy (500 μM) were incubated with ALP (0.1 or 1 U/mL) in PBS for different periods of time and quenched with equal volume of methanol. HPLC was used to determine the dephosphorylation of NBD-ffspy.

Determination of dephosphorylation by cells

In the conditioned media (CM)

Cells were seeded at 1.5×104 cells per well in a 96-well plate overnight to allow attachment. Cells were incubated with 100 μL NBD-ffspy (500 μM) for different periods of time, and their CM were collected, lyophilized, and then redissolved using methanol. HPLC was used to determine the dephosphorylation of NBD-ffspy in the conditioned media.

In the spheroids

Suspended HS-5 cells were seeded in NBD-ffspy (500 μM) containing culture media at 7.5×104 cells per dish in the inner 2 cm glass of a 3.5 cm confocal dish. Transfer CM and suspended cell spheroids and collect cell spheroids by centrifugation. Discard the supernatant and wash twice with PBS. The pellet was lyophilized and dissolved in methanol for LC-MS analysis.

Conditional dynamin I/II/III triple knockout of fibroblast

This protocol was developed by Pietro De Camilli’s group at Yale University School of Medicine (DOI: dx.doi.org/10.17504/protocols.io.b2ddqa26). Here we show it in detail for clarity. When cells reach 80-90% confluency, detach cells using trypsin-EDTA and split the cells 1:4. Add 2 μM 4-hydroxy-tamoxifen in fresh culture medium and incubate for 48 h. Split the cells 1:4 once again after 48 h. Add 300 nM 4-hydroxy-tamoxifen in fresh culture media and incubate for 72 h. Most of the dynamin disappears within the first 72-96 h but the full phenotype appears 120-144 h after starting the 4- hydroxy-tamoxifen treatment. KO efficiency is around 90%. Here, we seeded cells for experiments 120 h after the tamoxifen treatment. A dish of cells without 4- hydroxy-tamoxifen treatment is used as a control.

LAMP-1-RFP transduction

HS-5 cells were seeded in confocal dish with the addition of Lysosome-RFP at the concentration of 2 μL per 10,000 cells in DMEM and incubated for 24 h to allow attachment and transduction. Replace with fresh media containing NBD-ffspy (500 μM) and different exocytosis inhibitor for live cell imaging.

MTT assay

Cells were seeded at 104 cells per well in 96-well plates for 24 h to allow attachment. Culture media were replaced with fresh culture media supplemented with compounds at a series of concentrations. After 24/48/72 h, 10 μL of MTT solution (5 mg/mL) was added to each well and the plate was incubated in dark for 4 h at 37 °C. 100 μL of 10% SDS-HCl was added to each well to stop the reaction and dissolve the formazan. The absorbance of each well at 595 nm was determined by a microplate reader. The assay was performed three times to obtain the mean value of three measurements. For 7-day cytotoxicity, seed cells at 5,000 cells per well and replace with D-peptide-containing fresh medium every 3 days.

FRAP assay

FRAP was performed on a Zeiss LSM 880 confocal microscopy using a 40×/1.4 Oil objective. Five pre-bleach images were collected followed by photobleaching with 488-nm laser at 100% intensity within a circle region with the diameter of ~6 μm. Another region was imaged without photobleaching as an internal control. 512×512 pixel images were captured at 0.26 s intervals using a 488-nm laser at 1% intensity with the pinhole set at 390 μm. Images were collected until no further recovery was evident. The fluorescence recovery of the photobleached region was normalized and fitted into an exponential function.

Western Blot

Cells were cultured to reach 80-90% confluency, lysed with 500 μL lysis buffer (protease inhibitor cocktail added) per 10 cm dish on ice, sonicated for 10 s, and freeze-thawed for three cycles to collect cell lysates from various cell lines. The lysates were centrifuged at 15,294 x g for 10 min at 4 °C, and the supernatant was collected. The proteins in lysates were denatured by adding sample loading buffer and incubation at 95 °C for 5 min. The lysate samples were loaded on to precast gels for electrophoresis under 120 V for 45 min, followed by blotting to PVDF membrane under 100 V for 90 min in ice bath. Membranes were blocked with blocking buffer for 1 h at room temperature and incubated with primary antibody (1:1000 dilution) at 4 °C overnight. After washing with TBST three times for 5 min per time, secondary antibody was added at 1:5000 dilution for 1 h at room temperature. After washing with TBST for six times, chemiluminescent substrate was added and incubated for 1 min. Membranes were then scanned using a blot scanner. After scanning and washing the membranes with TBST, the antibodies on the membranes were stripped by incubation in stripping buffer for 20 min. The membranes were then washed with TBST and blocked with blocking buffer for 1 h at room temperature. The membranes were ready for the next round of primary antibody incubation.

Statistics and Reproducibility

All results are presented as the mean ± s.d. The sample sizes are indicated in the figure legends.

Experiments in Fig. 1c, 3a-c, 3l-m, 4e-f, 5d, 5g-h, 6n, Supplementary Figure 19 b-c, 20, 22d-e, 25a, 26a, 27b, 28e, 29b, 29e, 34c, 40c, 41b, and 41d were repeated twice taken from distinct samples. Experiments in Fig. 1f, 3d, 3a-j, 3n, 5a, 5d, 6a, 6e, 6j, 6m, 6o-p, Supplementary Figure 19a, 21a-b, 24a-b, 27a, 28b-d, 29a, 29c, 30a-b, 31a-f, 34a, 35, 36a-b, 37, 38 a-c, 40 a, 42a, and 43 were repeated three times taken from distinct samples. Experiment in Fig. 2a and 2b were repeated to obtain 7680 and 20808 micrographs of cryo-EM images, respectively.

Supplementary Material

Supporting information
Supplementary_Video_1
Download video file (5.6MB, avi)

Acknowledgements

This work was partially supported by NIH Grants CA142746 (B.X.), GM122510 (E.H.E.) GM138756 (F.W.), and NSF Grant DMR-2011846 (B.X.). This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract 75N91019D00024. The cryo-EM imaging was, in part, done at the Molecular Electron Microscopy Core Facility at the University of Virginia. The cryo-EM screening process was, in part, supported by the O’Neal Comprehensive Cancer Center at the University of Alabama Birmingham. We thank Prof. Pietro De Camilli for kindly providing the TKO cell line. We thank Dr. J. T. Hsien for kindly providing HeLa-GFP, PC-3-DsRed, and Saos-2-GFP.

Footnotes

Competing interests

The authors declare no competing interests.

Data availability

The cryo-EM models of structures reported in this study are deposited to Protein Data Bank (PDB) under the deposition ID 7T6E for class 1 NBD-ffsy filaments, 8DST for class 2 NBD-ffsy filaments, and 8FOF for BP-ffsy filaments. The data generated in this study are available in the article and Supplementary Information. Source data are provided with this paper. Source data is available for Figures 1, 3, 4, 5, and 6 and Supplementary Figures 22d, 34a and 40c in the associated supplementary Data 1, 2, 3, respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting information
NIHMS1903281-supplement-Supporting_information.pdf (7.8MB, pdf)
Supplementary_Video_1
Download video file (5.6MB, avi)

Data Availability Statement

The cryo-EM models of structures reported in this study are deposited to Protein Data Bank (PDB) under the deposition ID 7T6E for class 1 NBD-ffsy filaments, 8DST for class 2 NBD-ffsy filaments, and 8FOF for BP-ffsy filaments. The data generated in this study are available in the article and Supplementary Information. Source data are provided with this paper. Source data is available for Figures 1, 3, 4, 5, and 6 and Supplementary Figures 22d, 34a and 40c in the associated supplementary Data 1, 2, 3, respectively.

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