Cancer Immunology, Immunotherapy

, Volume 68, Issue 9, pp 1479–1492 | Cite as

An optimized retinoic acid-inducible gene I agonist M8 induces immunogenic cell death markers in human cancer cells and dendritic cell activation

  • Luciano CastielloEmail author
  • Alessandra Zevini
  • Elisabetta Vulpis
  • Michela Muscolini
  • Matteo Ferrari
  • Enrico Palermo
  • Giovanna Peruzzi
  • Christian Krapp
  • Martin Jakobsen
  • David Olagnier
  • Alessandra Zingoni
  • Angela Santoni
  • John HiscottEmail author
Original Article


RIG-I is a cytosolic RNA sensor that recognizes short 5′ triphosphate RNA, commonly generated during virus infection. Upon activation, RIG-I initiates antiviral immunity, and in some circumstances, induces cell death. Because of this dual capacity, RIG-I has emerged as a promising target for cancer immunotherapy. Previously, a sequence-optimized RIG-I agonist (termed M8) was generated and shown to stimulate a robust immune response capable of blocking viral infection and to function as an adjuvant in vaccination strategies. Here, we investigated the potential of M8 as an anti-cancer agent by analyzing its ability to induce cell death and activate the immune response. In multiple cancer cell lines, M8 treatment strongly activated caspase 3-dependent apoptosis, that relied on an intrinsic NOXA and PUMA-driven pathway that was dependent on IFN-I signaling. Additionally, cell death induced by M8 was characterized by the expression of markers of immunogenic cell death-related damage-associated molecular patterns (ICD-DAMP)—calreticulin, HMGB1 and ATP—and high levels of ICD-related cytokines CXCL10, IFNβ, CCL2 and CXCL1. Moreover, M8 increased the levels of HLA-ABC expression on the tumor cell surface, as well as up-regulation of genes involved in antigen processing and presentation. M8 induction of the RIG-I pathway in cancer cells favored dendritic cell phagocytosis and induction of co-stimulatory molecules CD80 and CD86, together with increased expression of IL12 and CXCL10. Altogether, these results highlight the potential of M8 in cancer immunotherapy, with the capacity to induce ICD-DAMP on tumor cells and activate immunostimulatory signals that synergize with current therapies.


RIG-I Interferons Cancer immunotherapy Immunogenic cell death Dendritic cells 



7-Aminoactinomycin D






Antigen processing machinery


American Type Culture Collection


Bcl-2-associated X protein


Becton Dickinson


Bcl-2 homology 3


Caspase recruitment domain


Carbonyl cyanide 3-chlorophenylhydrazone


C-C motif chemokine ligand 2


Cell trace far red


C-X-C motif ligand 1


C-X-C motif chemokine 10


Damage-associated molecular patterns


Epstein–Barr virus


Kaighn’s modification of Ham’s F-12 medium


Forward scatter-area


Forward scatter-height


Glyceraldehyde 3-phosphate dehydrogenase




4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid


Human leucocyte antigens-A, B, and C loci


High-mobility group box 1


Immunogenic cell death


Indoleamine-pyrrole 2,3-dioxygenase 1




Interferon α and β receptor subunit 1


Type-I interferon


IκB kinase


Interferon regulatory factor 3


Mitochondrial antiviral signaling protein


Myeloid-derived suppressor cells




Monocyte-derived dendritic cells


Penicillin–streptomycin solution


Pathogen-associated molecular patterns


Poly (ADP-ribose) polymerase


Pattern recognition receptors


Proteasome subunit beta type-8, -9, -10


p53 upregulated modulator of apoptosis


RIG-I agonist


Retinoic acid-inducible gene-I


Receptor-interacting protein 1


Relative light units


Short interfering RNA


Tumor-associated macrophages


Antigen peptide transporter 1, 2




TANK-binding kinase 1


Vesicular stomatitis virus




Author contributions

Conception and design: LC, AZ, JH, DO. Collection and assembly of data: LC, AZ, EV, MM, MF, EP, GP, CK. Data analysis and interpretation: LC, MJ, DO, AZ, AS, and JH. Manuscript writing: LC, AZ, JH. Final approval of manuscript: all authors.


This research was supported by grants from Fondazione Cenci Bolognetti, the Italian Association for Cancer Research (AIRC) (IG16901), and NIH Grants 1R561AI108861-01A1 and 7R21CA192185. Elisabetta Vulpis is supported by an AIRC fellowship for Italy.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

PBMC were freshly isolated from peripheral blood samples of anonymous volunteer healthy donors at the Transfusion Center of Sapienza University of Rome. Written informed consent was obtained from all blood donors to the use of their blood for scientific purposes.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of the Sapienza University of Rome (Rif.3373/250914) and with the 1964 Helsinki declaration. Institutional Review Board approval was not required for this kind of study.

Cell line authentication

Primary Mel1007 and metastatic melanoma Mel120 cells were a kind gift of Dr. G. Parmiani (Milan, Italy), A549, HCT116 and PC3 were all from ATCC. All the experiments were performed with cells at low passage numbers (≤ 10). Cell identity was monitored based on morphology and growth rate. Mycoplasma was routinely tested.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Luciano Castiello
    • 1
    • 5
    Email author
  • Alessandra Zevini
    • 1
  • Elisabetta Vulpis
    • 2
  • Michela Muscolini
    • 1
  • Matteo Ferrari
    • 1
  • Enrico Palermo
    • 1
  • Giovanna Peruzzi
    • 3
  • Christian Krapp
    • 4
  • Martin Jakobsen
    • 4
  • David Olagnier
    • 4
  • Alessandra Zingoni
    • 2
  • Angela Santoni
    • 1
    • 2
  • John Hiscott
    • 1
    Email author
  1. 1.Istituto Pasteur Italia-Cenci Bolognetti FoundationRomeItaly
  2. 2.Department of Molecular MedicineSapienza UniversityRomeItaly
  3. 3.Center for Life Nano Science@SapienzaIstituto Italiano di TecnologiaRomeItaly
  4. 4.Department of BiomedicineAarhus UniversityAarhusDenmark
  5. 5.FaBioCell, Core FacilitiesIstituto Superiore di SanitàRomeItaly

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