Article

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer

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Abstract

The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.

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Acknowledgements

This work was primarily supported by the Cancer Prevention and Research Institute of Texas, the Knowledge Gap funding of MD Anderson Cancer Center, and NCI grant CA213233. Other support include: V.S.L. laboratory: UT MDACC Khalifa Bin Zayed Al Nahya Foundation; High Resolution Electron Microscopy Facility: Institutional Core Grant CA16672; MDACC Flow Cytometry core facility; and J.J.L. laboratory: NIH P30CA16672; MDACC Small Animal Imaging Facility: NIH P30-CA016672 and 5U24-CA126577. The mass spectrometry-related analysis (SAM) was supported by the NORTE-01-0145-FEDER-000029 (NORTE 2020; ERDF) and FEDER funds (POCI-01/0145-FEDER-016618) and FCT-PTDC/BIM-ONC/2754/2014. We thank K. Dunner Jr for the help with the transmission electron microscopy and immunogold techniques, E. Chang for slide scanning, D. Lundy for tissue processing, J. Carstens and C. Kahlert for independent analyses of tissue histopathology, J. Kaye and L. Gibson for mouse husbandry support, C. Kingsley and D. Lundy for MRI support, E. Lawson for IVIS imaging help, and P. Correa-de-Sampaio for microscopy imaging help.

Author information

Affiliations

  1. Department of Cancer Biology, Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston, Texas 77005, USA

    • Sushrut Kamerkar
    • , Valerie S. LeBleu
    • , Hikaru Sugimoto
    • , Sujuan Yang
    • , Sonia A. Melo
    •  & Raghu Kalluri
  2. Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal (I3S), 4200 Porto, Portugal; Institute of Pathology and Molecular Immunology of the University of Porto (IPATIMUP), 4200 Porto, Portugal

    • Carolina F. Ruivo
    •  & Sonia A. Melo
  3. Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, Texas 77005, USA

    • J. Jack Lee

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Contributions

R.K. conceptually designed the experimental strategy, provided intellectual input and helped to write the manuscript. H.S. and V.S.L. injected mice orthotopically with tumour cells. H.S. performed necropsy analyses and quantification of exosomes in pancreas tissue sections. V.S.L. provided intellectual input, extracted RNA and quantified siRNA loading in exosomes by qPCR analyses, stained tissues for Kras, helped with in vivo experiments, designed the experimental strategy, prepared figures and wrote the manuscript. S.K. and S.Y. prepared exosomes (cultured cells for exosome collection and preparation by ultracentrifugation), generated iExosomes (electroporation and wash steps involved in the generation of iExosome and NanoSight measurements), and treated mice with iExosomes. S.Y. also performed an independent analysis of in vivo imaging data, generated and genotyped KTC and KPC mice for iExosome treatment, and helped prepare the figures. S.K. provided intellectual input, helped design the experimental strategy, and performed experiments, including treatment of mice with iExosomes, preparation of exosomes for sucrose gradient and qPCR analyses. Unless otherwise noted, S.K. performed the independent replication of the experiments. S.K. performed sucrose gradient analyses, NanoSight measurements, flow cytometry experiments and analyses, immunostaining and analyses, RNA extraction and qPCR of treated cells, MTT and TUNEL assays, macropinocytosis and western blot analyses, analysed data, prepared figures, and helped to write the manuscript. S.A.M. advised and provided intellectual input to the siRNA identification experiments, MTT assays, qPCR analyses, western blots for p-ERK, and one PANC-1 tumour study and one KTC in vivo experiment. C.F.R. provided the mass spectrometry data and the relevant section in the Methods. J.J.L. reviewed the source data, advised on experimental design, and supervised statistical analyses.

Competing interests

MD Anderson Cancer Center and R.K. hold patents in the area of exosome biology and are licensed to Codiak Biosciences Inc. MD Anderson Cancer Center and R.K. are stock equity holders in Codiak Biosciences Inc. R.K. receives research support from Codiak Biosciences Inc. and serves as a member of the board of directors. V.S.L. served once as a paid consultant for Codiak Biosciences Inc.

Corresponding author

Correspondence to Raghu Kalluri.

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Extended data

Supplementary information

PDF files

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    Supplementary Information

    This file contains Supplementary Text regarding the Results sections, a Supplementary Discussion, Supplementary details regarding the figure legends, Supplementary References and Supplementary Figure 1, the uncropped blots.

Excel files

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    Supplementary Table 1

    This file contains results from Mass Spectrometry analysis of exosomes from T3M4 exosomes from CD24+CD44+ cells, see the Methods section for details.

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