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:

Targeting Xkr8 via nanoparticle-mediated in situ co-delivery of siRNA and chemotherapy drugs for cancer immunochemotherapy

Abstract

Activation of scramblases is one of the mechanisms that regulates the exposure of phosphatidylserine to the cell surface, a process that plays an important role in tumour immunosuppression. Here we show that chemotherapeutic agents induce overexpression of Xkr8, a scramblase activated during apoptosis, at the transcriptional level in cancer cells, both in vitro and in vivo. Based on this finding, we developed a nanocarrier for co-delivery of Xkr8 short interfering RNA and the FuOXP prodrug to tumours. Intravenous injection of our nanocarrier led to significant inhibition of tumour growth in colon and pancreatic cancer models along with increased antitumour immune response. Targeting Xkr8 in combination with chemotherapy may represent a novel strategy for the treatment of various types of cancers.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Fig. 1: Xkr8 was induced by chemotherapeutic agents in vitro and in vivo.
Fig. 2: Development and biophysical characterization of PMBOP-CP-based nanocarrier for co-delivery of siXkr8 and FuOXP.
Fig. 3: Optimizing PMBOP-CP NPs for effective tumour targeting in vivo.
Fig. 4: CD44-mediated vascular targeting plays a role in tumour targeting.
Fig. 5: In vivo PK and tissue distribution of siXkr8 and FuOXP following intravenous administration of FuOXP/siXkr8 NPs, and the efficiency of gene knockdown.
Fig. 6: Biological consequences of Xkr8 knockdown in vitro and in vivo.

Similar content being viewed by others

Data availability

The bulk messenger RNA-seq data mapped to the mouse genome (GRCm38: https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/) are available in the NCBI Gene Expression Omnibus (GEO) under accession number GSE214881 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE214881). Source data are provided with this paper. All data generated or analysed during this study are included in this Article and its Supplementary Information files.

References

  1. Birge, R. B. et al. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ. 23, 962–978 (2016).

    Article  CAS  Google Scholar 

  2. Kumar, S., Calianese, D. & Birge, R. B. Efferocytosis of dying cells differentially modulate immunological outcomes in tumor microenvironment. Immunol. Rev. 280, 149–164 (2017).

    Article  CAS  Google Scholar 

  3. Nagata, S., Suzuki, J., Segawa, K. & Fujii, T. Exposure of phosphatidylserine on the cell surface. Cell Death Differ. 23, 952–961 (2016).

    Article  CAS  Google Scholar 

  4. Hankins, H. M., Baldridge, R. D., Xu, P. & Graham, T. R. Role of flippases, scramblases and transfer proteins in phosphatidylserine subcellular distribution. Traffic 16, 35–47 (2015).

    Article  CAS  Google Scholar 

  5. Suzuki, J., Denning, D. P., Imanishi, E., Horvitz, H. R. & Nagata, S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341, 403–406 (2013).

    Article  CAS  Google Scholar 

  6. Suzuki, J., Imanishi, E. & Nagata, S. Xkr8 phospholipid scrambling complex in apoptotic phosphatidylserine exposure. Proc. Natl Acad. Sci. USA 113, 9509–9514 (2016).

    Article  CAS  Google Scholar 

  7. Huang, Q. et al. Caspase 3-mediated stimulation of tumor cell repopulation during cancer radiotherapy. Nat. Med. 17, 860–866 (2011).

    Article  CAS  Google Scholar 

  8. Sakuragi, T., Kosako, H. & Nagata, S. Phosphorylation-mediated activation of mouse Xkr8 scramblase for phosphatidylserine exposure. Proc. Natl Acad. Sci. USA 116, 2907–2912 (2019).

    Article  CAS  Google Scholar 

  9. Ravichandran, K. S. Find-me and eat-me signals in apoptotic cell clearance: progress and conundrums. J. Exp. Med. 207, 1807–1817 (2010).

    Article  CAS  Google Scholar 

  10. Hochreiter-Hufford, A. & Ravichandran, K. S. Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb. Perspect. Biol. https://doi.org/10.1101/cshperspect.a008748 (2013).

  11. Kang, T. H. et al. Annexin A5 as an immune checkpoint inhibitor and tumor-homing molecule for cancer treatment. Nat. Commun. https://doi.org/10.1038/s41467-020-14821-z (2020).

  12. Chang, W., Fa, H., Xiao, D. & Wang, J. Targeting phosphatidylserine for cancer therapy: prospects and challenges. Theranostics 10, 9214–9229 (2020).

    Article  CAS  Google Scholar 

  13. Thorpe, P. E. Targeting anionic phospholipids on tumor blood vessels and tumor cells. Thromb. Res. 125, S134–S137 (2010).

    Article  Google Scholar 

  14. Sun, A. & Benet, L. Z. Late-stage failures of monoclonal antibody drugs: a retrospective case study analysis. Pharmacology 105, 145–163 (2020).

    Article  CAS  Google Scholar 

  15. Shin, S. A., Moon, S. Y., Park, D., Park, J. B. & Lee, C. S. Apoptotic cell clearance in the tumor microenvironment: a potential cancer therapeutic target. Arch. Pharm. Res 42, 658–671 (2019).

    Article  CAS  Google Scholar 

  16. Zhang, R., Song, X.-Q., Liu, R.-P., Ma, Z.-Y. & Xu, J.-Y. Fuplatin: an efficient and low-toxic dual-prodrug. J. Med. Chem. 62, 4543–4554 (2019).

    Article  CAS  Google Scholar 

  17. Li, M., Schlesiger, S., Knauer, S. K. & Schmuck, C. A tailor-made specific anion-binding motif in the side chain transforms a tetrapeptide into an efficient vector for gene delivery. Angew. Chem. 127, 2984–2987 (2015).

    Article  Google Scholar 

  18. Mitchell, M. J. et al. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 20, 101–124 (2021).

    Article  CAS  Google Scholar 

  19. Li, M. et al. Drug delivery systems based on CD44-targeted glycosaminoglycans for cancer therapy. Carbohydr. Polym. 251, 117103 (2021).

    Article  CAS  Google Scholar 

  20. Mattheolabakis, G., Milane, L., Singh, A. & Amiji, M. M. Hyaluronic acid targeting of CD44 for cancer therapy: from receptor biology to nanomedicine. J. Drug Target 23, 605–618 (2015).

    Article  CAS  Google Scholar 

  21. Luo, Z., Dai, Y. & Gao, H. Development and application of hyaluronic acid in tumor targeting drug delivery. Acta Pharm. Sin. B 9, 1099–1112 (2019).

    Article  Google Scholar 

  22. Qhattal, H. S., Hye, T., Alali, A. & Liu, X. Hyaluronan polymer length, grafting density, and surface poly(ethylene glycol) coating influence in vivo circulation and tumor targeting of hyaluronan-grafted liposomes. ACS Nano 8, 5423–5440 (2014).

    Article  CAS  Google Scholar 

  23. Teng, C. et al. Desirable PEGylation for improving tumor selectivity of hyaluronic acid-based nanoparticles via low hepatic captured, long circulation times and CD44 receptor-mediated tumor targeting. Nanomedicine 24, 102105 (2020).

    Article  CAS  Google Scholar 

  24. Subhan, M. A., Yalamarty, S. S. K., Filipczak, N., Parveen, F. & Torchilin, V. P. Recent advances in tumor targeting via EPR effect for cancer treatment. J. Pers Med. https://doi.org/10.3390/jpm11060571 (2021).

  25. Sindhwani, S. et al. The entry of nanoparticles into solid tumours. Nat. Mater. 19, 566–575 (2020).

    Article  CAS  Google Scholar 

  26. Griffioen, A. W. et al. CD44 is involved in tumor angiogenesis; an activation antigen on human endothelial cells. Blood 90, 1150–1159 (1997).

    Article  CAS  Google Scholar 

  27. Auerbach, R., Akhtar, N., Lewis, R. L. & Shinners, B. L. Angiogenesis assays: problems and pitfalls. Cancer Metastasis Rev. 19, 167–172 (2000).

    Article  CAS  Google Scholar 

  28. Vojtek, M. et al. Fast and reliable ICP-MS quantification of palladium and platinum-based drugs in animal pharmacokinetic and biodistribution studies. Anal. Methods 12, 4806–4812 (2020).

    Article  CAS  Google Scholar 

  29. Kumar, V. et al. Pharmacokinetics and biodistribution of polymeric micelles containing miRNA and small-molecule drug in orthotopic pancreatic tumor-bearing mice. Theranostics 8, 4033–4049 (2018).

    Article  CAS  Google Scholar 

  30. Wang, H. & Guo, P. Radiolabeled RNA nanoparticles for highly specific targeting and efficient tumor accumulation with favorable in vivo biodistribution. Mol. Pharm. 18, 2924–2934 (2021).

    Article  CAS  Google Scholar 

  31. Vocelle, D., Chan, C. & Walton, S. P. Endocytosis controls siRNA efficiency: implications for siRNA delivery vehicle design and cell-specific targeting. Nucleic Acid Ther. 30, 22–32 (2020).

    Article  CAS  Google Scholar 

  32. Dong, Y., Siegwart, D. J. & Anderson, D. G. Strategies, design, and chemistry in siRNA delivery systems. Adv. Drug Deliv. Rev. 144, 133–147 (2019).

    Article  CAS  Google Scholar 

  33. Song, W. et al. Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap. Nat. Commun. 9, 2237 (2018).

    Article  Google Scholar 

  34. Lima, L. G., Chammas, R., Monteiro, R. Q., Moreira, M. E. & Barcinski, M. A. Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner. Cancer Lett. 283, 168–175 (2009).

    Article  CAS  Google Scholar 

  35. Sharma, R., Huang, X., Brekken, R. A. & Schroit, A. J. Detection of phosphatidylserine-positive exosomes for the diagnosis of early-stage malignancies. Br. J. Cancer 117, 545–552 (2017).

    Article  CAS  Google Scholar 

  36. Proto, J. D. et al. Regulatory T cells promote macrophage efferocytosis during inflammation resolution. Immunity 49, 666–677 e666 (2018).

    Article  CAS  Google Scholar 

  37. Qi, L. et al. IL-10 secreted by M2 macrophage promoted tumorigenesis through interaction with JAK2 in glioma. Oncotarget 7, 71673–71685 (2016).

    Article  Google Scholar 

  38. Gray, M. J. et al. Phosphatidylserine-targeting antibodies augment the anti-tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro-oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers. Breast Cancer Res. 18, 50 (2016).

    Article  Google Scholar 

  39. Snyder, A. G. et al. Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Sci. Immunol. https://doi.org/10.1126/sciimmunol.aaw2004 (2019).

  40. Liu, Y., Hardie, J., Zhang, X. & Rotello, V. M. Effects of engineered nanoparticles on the innate immune system. Semin. Immunol. 34, 25–32 (2017).

    Article  CAS  Google Scholar 

  41. Kawano, M. & Nagata, S. Lupus-like autoimmune disease caused by a lack of Xkr8, a caspase-dependent phospholipid scramblase. Proc. Natl Acad. Sci. USA 115, 2132–2137 (2018).

    Article  CAS  Google Scholar 

  42. Li, S. et al. Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors. Am. J. Physiol. 276, 796–804 (1999).

    Google Scholar 

  43. Chen, Y. et al. An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy. Nat. Commun. 7, 1–12 (2016).

    Article  Google Scholar 

  44. Shum, K. & Rossi, J. J. SiRNA Delivery Methods: Methods and Protocols (Humana Press, 2016).

  45. Sun, J. et al. A prodrug micellar carrier assembled from polymers with pendant farnesyl thiosalicylic acid moieties for improved delivery of paclitaxel. Acta Biomater. 43, 282–291 (2016).

    Article  CAS  Google Scholar 

  46. Tseng, W., Leong, X. & Engleman, E. Orthotopic mouse model of colorectal cancer. J. Vis. Exp. https://doi.org/10.3791/484 (2007).

  47. Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P. & Johnson, J. M. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737–1744 (2005).

    Article  CAS  Google Scholar 

  48. Lynch, R. W. et al. An efficient method to isolate Kupffer cells eliminating endothelial cell contamination and selective bias. J. Leukoc. Biol. 104, 579–586 (2018).

    Article  CAS  Google Scholar 

  49. Gorgun, C. et al. Isolation and flow cytometry characterization of extracellular-vesicle subpopulations derived from human mesenchymal stromal cells. Curr. Protoc. Stem Cell Biol. 48, e76 (2019).

    Article  Google Scholar 

  50. Ray, A. & Dittel, B. N. Isolation of mouse peritoneal cavity cells. J. Vis. Exp. https://doi.org/10.3791/1488 (2010).

  51. Horuluoglu, B. H., Kayraklioglu, N., Tross, D. & Klinman, D. PAM3 protects against DSS-induced colitis by altering the M2:M1 ratio. Sci. Rep. 10, 6078 (2020).

    Article  CAS  Google Scholar 

  52. Turnis, M. E. et al. Interleukin-35 limits anti-tumor immunity. Immunity 44, 316–329 (2016).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by National Institute of Health grants R01CA219399, R01CA223788 (to S.L.), R01CA219716 (to B.L. and S.L.) and a grant from the Shear Family Foundation. We thank R. Gibbs for his advice on statistical analysis.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization, Y.C., Y.H. and S.L. Methodology, Y.C. and Y.H. Validation, Y.C., Y.H., Q.L., Z.L., Z.Z. and H.H. Formal analysis, Y.C. Investigation, Y.C., Y.H., Q.L., Z.L., Z.Z., H.H., J.S., L.Z., R.S., D.B., J.F.C., B.L. and S.L. Visualization, Y.C. and Y.H. Writing—original draft, Y.C. and S.L. Writing—review and editing, Y.C. and S.L. Project administration, Y.C. and S.L. Funding acquisition, S.L. Resources, S.L. Supervision, S.L. and B.L.

Corresponding authors

Correspondence to Binfeng Lu or Song Li.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Nanotechnology thanks Shigekazu Nagata, David Oupicky 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

Table of contents, Supplementary methods, Supplementary Figs. 1–24, Table 2 and references.

Reporting Summary

Supplementary Table 1

List of siRNA and primer sequences.

Source data

Source Data For Fig. 1

Unprocessed Western Blots for Fig. 1.

Source Data For Fig. 2

Unprocessed gels for Fig. 2.

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

Chen, Y., Huang, Y., Li, Q. et al. Targeting Xkr8 via nanoparticle-mediated in situ co-delivery of siRNA and chemotherapy drugs for cancer immunochemotherapy. Nat. Nanotechnol. 18, 193–204 (2023). https://doi.org/10.1038/s41565-022-01266-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-022-01266-2

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research