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.
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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.
Birge, R. B. et al. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ. 23, 962–978 (2016).
Kumar, S., Calianese, D. & Birge, R. B. Efferocytosis of dying cells differentially modulate immunological outcomes in tumor microenvironment. Immunol. Rev. 280, 149–164 (2017).
Nagata, S., Suzuki, J., Segawa, K. & Fujii, T. Exposure of phosphatidylserine on the cell surface. Cell Death Differ. 23, 952–961 (2016).
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).
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).
Suzuki, J., Imanishi, E. & Nagata, S. Xkr8 phospholipid scrambling complex in apoptotic phosphatidylserine exposure. Proc. Natl Acad. Sci. USA 113, 9509–9514 (2016).
Huang, Q. et al. Caspase 3-mediated stimulation of tumor cell repopulation during cancer radiotherapy. Nat. Med. 17, 860–866 (2011).
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).
Ravichandran, K. S. Find-me and eat-me signals in apoptotic cell clearance: progress and conundrums. J. Exp. Med. 207, 1807–1817 (2010).
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).
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).
Chang, W., Fa, H., Xiao, D. & Wang, J. Targeting phosphatidylserine for cancer therapy: prospects and challenges. Theranostics 10, 9214–9229 (2020).
Thorpe, P. E. Targeting anionic phospholipids on tumor blood vessels and tumor cells. Thromb. Res. 125, S134–S137 (2010).
Sun, A. & Benet, L. Z. Late-stage failures of monoclonal antibody drugs: a retrospective case study analysis. Pharmacology 105, 145–163 (2020).
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).
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).
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).
Mitchell, M. J. et al. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 20, 101–124 (2021).
Li, M. et al. Drug delivery systems based on CD44-targeted glycosaminoglycans for cancer therapy. Carbohydr. Polym. 251, 117103 (2021).
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).
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).
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).
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).
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).
Sindhwani, S. et al. The entry of nanoparticles into solid tumours. Nat. Mater. 19, 566–575 (2020).
Griffioen, A. W. et al. CD44 is involved in tumor angiogenesis; an activation antigen on human endothelial cells. Blood 90, 1150–1159 (1997).
Auerbach, R., Akhtar, N., Lewis, R. L. & Shinners, B. L. Angiogenesis assays: problems and pitfalls. Cancer Metastasis Rev. 19, 167–172 (2000).
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).
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).
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).
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).
Dong, Y., Siegwart, D. J. & Anderson, D. G. Strategies, design, and chemistry in siRNA delivery systems. Adv. Drug Deliv. Rev. 144, 133–147 (2019).
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).
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).
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).
Proto, J. D. et al. Regulatory T cells promote macrophage efferocytosis during inflammation resolution. Immunity 49, 666–677 e666 (2018).
Qi, L. et al. IL-10 secreted by M2 macrophage promoted tumorigenesis through interaction with JAK2 in glioma. Oncotarget 7, 71673–71685 (2016).
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).
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).
Liu, Y., Hardie, J., Zhang, X. & Rotello, V. M. Effects of engineered nanoparticles on the innate immune system. Semin. Immunol. 34, 25–32 (2017).
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).
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).
Chen, Y. et al. An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy. Nat. Commun. 7, 1–12 (2016).
Shum, K. & Rossi, J. J. SiRNA Delivery Methods: Methods and Protocols (Humana Press, 2016).
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).
Tseng, W., Leong, X. & Engleman, E. Orthotopic mouse model of colorectal cancer. J. Vis. Exp. https://doi.org/10.3791/484 (2007).
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).
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).
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).
Ray, A. & Dittel, B. N. Isolation of mouse peritoneal cavity cells. J. Vis. Exp. https://doi.org/10.3791/1488 (2010).
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).
Turnis, M. E. et al. Interleukin-35 limits anti-tumor immunity. Immunity 44, 316–329 (2016).
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.
The authors declare no competing interests.
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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. (2022). https://doi.org/10.1038/s41565-022-01266-2