Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cell spheroid creation by transcytotic intercellular gelation

Nature Nanotechnology volume 18, pages 1094–1104 (2023)Cite this article

Subjects

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 cryogenic electron microscopy to determine the atomic structure of helical nanofibres self-assembled from enzyme-responsive d-peptides and fluorescent imaging to show that the transcytosis of d-peptides induces intercellular nanofibres/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 nanofibres. On secretion to the cell surface, these nanofibres form intercellular gels that act as artificial matrices and facilitate the fibrillogenesis of fibronectins to induce cell spheroids. No spheroid formation occurs without endo- or exocytosis, phosphate triggers or 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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Self-assembled d-peptide nanofibres form intercellular hydrogels.
Fig. 2: NBD–ffsy and BP–ffsy self-assemble into cross-β filaments in vitro.
Fig. 3: Spheroids from adherent and suspended HS-5 cells consist of intercellular hydrogel that colocalizes with fibronectin.
Fig. 4: Endocytosis and exocytosis are crucial for spheroid formation.
Fig. 5: Intracellular dephosphorylation is crucial for spheroid formation.
Fig. 6: d-peptide nanofibres facilitate the fibrillogenesis of fibronectin.

Similar content being viewed by others

Data availability

The cryo-EM models of the structures reported in this study are deposited in the Protein Data Bank (PDB) under deposition ID 7L17 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 its Supplementary Information. Source data are provided with this paper.

References

  1. Rookmaaker, M. B., Schutgens, F., Verhaar, M. C. & Clevers, H. Development and application of human adult stem or progenitor cell organoids. Nat. Rev. Nephrol. 11, 546–554 (2015).

    Article  CAS  Google Scholar 

  2. Baharvand, H., Hashemi, S. M., Kazemi Ashtiani, S. & Farrokhi, A. Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro. Int. J. Dev. Biol. 50, 645–652 (2006).

    Article  CAS  Google Scholar 

  3. Benya, P. D. & Shaffer, J. D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30, 215–224 (1982).

    Article  CAS  Google Scholar 

  4. Nelson, C. M. & Bissell, M. J. Modeling dynamic reciprocity: engineering three-dimensional culture models of breast architecture, function, and neoplastic transformation. Semin. Cancer Biol. 15, 342–352 (2005).

    Article  Google Scholar 

  5. Imamura, Y. et al. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncol. Rep. 33, 1837–1843 (2015).

    Article  CAS  Google Scholar 

  6. Weaver, V. M. et al. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J. Cell Biol. 137, 231–245 (1997).

    Article  CAS  Google Scholar 

  7. Bhadriraju, K. & Chen, C. S. Engineering cellular microenvironments to improve cell-based drug testing. Drug Discov. Today 7, 612–620 (2002).

    Article  CAS  Google Scholar 

  8. Yamada, K. M. & Cukierman, E. Modeling tissue morphogenesis and cancer in 3D. Cell 130, 601–610 (2007).

    Article  CAS  Google Scholar 

  9. Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science 294, 1708–1712 (2001).

    Article  CAS  Google Scholar 

  10. Gao, D. et al. Organoid cultures derived from patients with advanced prostate cancer. Cell 159, 176–187 (2014).

    Article  CAS  Google Scholar 

  11. Karthaus, W. R. et al. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 159, 163–175 (2014).

    Article  CAS  Google Scholar 

  12. Drost, J. et al. Organoid culture systems for prostate epithelial and cancer tissue. Nat. Protoc. 11, 347–358 (2016).

    Article  CAS  Google Scholar 

  13. Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M. & Nielsen, L. K. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol. Bioeng. 83, 173–180 (2003).

    Article  CAS  Google Scholar 

  14. Zheng, H. et al. Rotary culture promotes the proliferation of MCF-7 cells encapsulated in three-dimensional collagen–alginate hydrogels via activation of the ERK1/2-MAPK pathway. Biomed. Mater. 7, 015003 (2012).

    Article  Google Scholar 

  15. Raghavan, S. et al. Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity. Oncotarget 7, 16948–16961 (2016).

    Article  Google Scholar 

  16. Marchi, F. & Leblond, C. P. Collagen biogenesis and assembly into fibrils as shown by ultrastructural and 3H-proline radioautographic studies on the fibroblasts of the rat food pad. Am. J. Anat. 168, 167–197 (1983).

    Article  CAS  Google Scholar 

  17. McCaffrey, G. et al. Tight junctions contain oligomeric protein assembly critical for maintaining blood–brain barrier integrity in vivo. J. Neurochem. 103, 2540–2555 (2007).

    CAS  Google Scholar 

  18. Seger, D., Seger, R. & Shaltiel, S. The CK2 phosphorylation of vitronectin. Promotion of cell adhesion via the αvβ3-phosphatidylinositol 3-kinase pathway. J. Biol. Chem. 276, 16998–17006 (2001).

    Article  CAS  Google Scholar 

  19. Weber, G. F. et al. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. J. Leukoc. Biol. 72, 752–761 (2002).

    Article  CAS  Google Scholar 

  20. Yalak, G. & Vogel, V. Extracellular phosphorylation and phosphorylated proteins: not just curiosities but physiologically important. Sci. Signal. 5, re7 (2012).

    Article  Google Scholar 

  21. Wu, D. et al. Polymers with controlled assembly and rigidity made with click-functional peptide bundles. Nature 574, 658–662 (2019).

    Article  CAS  Google Scholar 

  22. Du, X., Zhou, J., Shi, J. & Xu, B. Supramolecular hydrogelators and hydrogels: from soft matter to molecular biomaterials. Chem. Rev. 115, 13165–13307 (2015).

    Article  CAS  Google Scholar 

  23. Cheetham, A. G., Zhang, P., Lin, Y. A., Lock, L. L. & Cui, H. Supramolecular nanostructures formed by anticancer drug assembly. J. Am. Chem. Soc. 135, 2907–2910 (2013).

    Article  CAS  Google Scholar 

  24. Tibbitt, M. W. & Anseth, K. S. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng. 103, 655–663 (2009).

    Article  CAS  Google Scholar 

  25. Jayawarna, V. et al. Nanostructured hydrogels for three‐dimensional cell culture through self‐assembly of fluorenylmethoxycarbonyl–dipeptides. Adv. Mater. 18, 611–614 (2006).

    Article  CAS  Google Scholar 

  26. Smith, D. J. et al. A multiphase transitioning peptide hydrogel for suturing ultrasmall vessels. Nat. Nanotechnol. 11, 95–102 (2016).

    Article  CAS  Google Scholar 

  27. Alvarez, Z. et al. Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury. Science 374, 848–856 (2021).

    Article  CAS  Google Scholar 

  28. Winkler, S. M., Harrison, M. R. & Messersmith, P. B. Biomaterials in fetal surgery. Biomater. Sci. 7, 3092–3109 (2019).

    Article  CAS  Google Scholar 

  29. Wang, H., Feng, Z. & Xu, B. Intercellular instructed-assembly mimics protein dynamics to induce cell spheroids. J. Am. Chem. Soc. 141, 7271–7274 (2019).

    Article  CAS  Google Scholar 

  30. Wang, H. et al. An in situ dynamic continuum of supramolecular phosphoglycopeptides enables formation of 3D cell spheroids. Angew. Chem. Int. Ed. 56, 16297–16301 (2017).

    Article  CAS  Google Scholar 

  31. He, H. et al. Enzymatic noncovalent synthesis. Chem. Rev. 120, 9994–10078 (2020).

    Article  CAS  Google Scholar 

  32. Zhang, Y., Kuang, Y., Gao, Y. & Xu, B. Versatile small-molecule motifs for self-assembly in water and the formation of biofunctional supramolecular hydrogels. Langmuir 27, 529–537 (2011).

    Article  CAS  Google Scholar 

  33. Reches, M. & Gazit, E. Casting metal nanowires within discrete self-assembled peptide nanotubes. Science 300, 625–627 (2003).

    Article  Google Scholar 

  34. Gao, Y., Shi, J., Yuan, D. & Xu, B. Imaging enzyme-triggered self-assembly of small molecules inside live cells. Nat. Commun. 3, 1033 (2012).

    Article  Google Scholar 

  35. Van Itallie, C. M. & Anderson, J. M. Occludin confers adhesiveness when expressed in fibroblasts. J. Cell Sci. 110, 1113–1121 (1997).

    Article  Google Scholar 

  36. Mrsny, R. J. et al. A key claudin extracellular loop domain is critical for epithelial barrier integrity. Am. J. Pathol. 172, 905–915 (2008).

    Article  CAS  Google Scholar 

  37. Lee, M., Ghosh, U., Thurber, K. R., Kato, M. & Tycko, R. Molecular structure and interactions within amyloid-like fibrils formed by a low-complexity protein sequence from FUS. Nat. Commun. 11, 5735 (2020).

    Article  Google Scholar 

  38. Roder, C. et al. Atomic structure of PI3-kinase SH3 amyloid fibrils by cryo-electron microscopy. Nat. Commun. 10, 3754 (2019).

    Article  Google Scholar 

  39. Cao, Q., Boyer, D. R., Sawaya, M. R., Ge, P. & Eisenberg, D. S. Cryo-EM structures of four polymorphic TDP-43 amyloid cores. Nat. Struct. Mol. Biol. 26, 619–627 (2019).

    Article  CAS  Google Scholar 

  40. Fitzpatrick, A. W. P. et al. Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547, 185–190 (2017).

    Article  CAS  Google Scholar 

  41. Falcon, B. et al. Novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules. Nature 568, 420–423 (2019).

    Article  CAS  Google Scholar 

  42. Wang, F. et al. Deterministic chaos in the self-assembly of β sheet nanotubes from an amphipathic oligopeptide. Matter 4, 3217–3231 (2021).

  43. To, W. S. & Midwood, K. S. Plasma and cellular fibronectin: distinct and independent functions during tissue repair. Fibrogenesis Tissue Repair 4, 21 (2011).

    Article  CAS  Google Scholar 

  44. Lu, Y. et al. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A. Autophagy 10, 1895–1905 (2014).

    Article  Google Scholar 

  45. Du, X. et al. In situ generated d‐peptidic nanofibrils as multifaceted apoptotic inducers to target cancer cells. Cell Death Dis. 8, e2614–e2614 (2017).

    Article  CAS  Google Scholar 

  46. Feng, Z., Wang, H., Chen, X. & Xu, B. Self-assembling ability determines the activity of enzyme-instructed self-assembly for inhibiting cancer cells. J. Am. Chem. Soc. 139, 15377–15384 (2017).

    Article  CAS  Google Scholar 

  47. Shigemitsu, H. et al. An adaptive supramolecular hydrogel comprising self-sorting double nanofibre networks. Nat. Nanotechnol. 13, 165–172 (2018).

    Article  CAS  Google Scholar 

  48. Epstein, I. R. & Xu, B. Reaction–diffusion processes at the nano- and microscales. Nat. Nanotechnol. 11, 312–319 (2016).

    Article  CAS  Google Scholar 

  49. Liang, G., Ren, H. & Rao, J. A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2, 54–60 (2010).

    Article  CAS  Google Scholar 

  50. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

  51. Ottinger, E. A., Shekels, L. L., Bernlohr, D. A. & Barany, G. Synthesis of phosphotyrosine-containing peptides and their use as substrates for protein tyrosine phosphatases. Biochemistry 32, 4354–4361 (1993).

    Article  CAS  Google Scholar 

  52. Liu, S. et al. Enzymatically forming intranuclear peptide assemblies for selectively killing human induced pluripotent stem cells. J. Am. Chem. Soc. 143, 15852–15862 (2021).

    Article  CAS  Google Scholar 

  53. Basu Ray, G., Chakraborty, I. & Moulik, S. P. Pyrene absorption can be a convenient method for probing critical micellar concentration (cmc) and indexing micellar polarity. J. Colloid Interface Sci. 294, 248–254 (2006).

    Article  CAS  Google Scholar 

  54. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  55. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  56. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  Google Scholar 

  57. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  58. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was partially supported by National Institutes of Health (NIH) grants CA142746 (B.X.), GM122510 (E.H.E.) and GM138756 (F.W.), as well as 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 at Birmingham. We thank P. D. Camilli for providing the TKO cell line. We thank J. T. Hsien for providing HeLa–GFP, PC-3–DsRed and Saos-2–GFP.

Author information

Authors and Affiliations

  1. Department of Chemistry, Brandeis University, Waltham, MA, USA

    Jiaqi Guo, Yimeng Huang, Hongjian He, Weiyi Tan, Meihui Yi & Bing Xu

  2. Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA

    Fengbin Wang & Edward H. Egelman

  3. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA

    Fengbin Wang

  4. O’Neal Comprehensive Cancer Center University of Alabama at Birmingham, Birmingham, AL, USA

    Fengbin Wang

Authors
  1. Jiaqi Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fengbin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yimeng Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hongjian He
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Weiyi Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Meihui Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Edward H. Egelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bing Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.X. and J.G. conceived the study. J.G., under the supervision of B.X., designed and performed the chemical synthesis, generated the images in cell-free and cell-based assays and analysed the results. Y.H. and M.Y. assisted in the chemical synthesis. H.H. and W.T. performed the liquid chromatography–mass spectrometry analysis. F.W. and E.H.E. performed the cryo-EM reconstructions and model building. J.G., F.W., E.H.E. and B.X. wrote the manuscript with inputs from all authors.

Corresponding authors

Correspondence to Fengbin Wang, Edward H. Egelman or Bing Xu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Nanotechnology thanks Deling Kong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–43, Tables 1 and 2, materials and instruments and references.

Reporting Summary

Supplementary Video 1

Suspended HS-5 cells incubated with NBD–ffspy (500 µM) over the first 6 h.

Supplementary Data 1

Full scans of the western blot results in Supplementary Fig. 22d.

Supplementary Data 2

Full scans of the western blot results in Supplementary Fig. 34a.

Supplementary Data 3

Full scans of the western blot results in Supplementary Fig. 40c.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guo, J., Wang, F., Huang, Y. et al. Cell spheroid creation by transcytotic intercellular gelation. Nat. Nanotechnol. 18, 1094–1104 (2023). https://doi.org/10.1038/s41565-023-01401-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-023-01401-7

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing