Category: Preclinical Development
Purpose: We previously synthesized a docosahexaenoyl difluorodeoxycytidine amide (DHA-dFdC) by conjugating docosahexaenoic acid (DHA) to the 4-N position on 2’,2’-difluorodeoxycytidine (dFdC). DHA-dFdC shows potent, broad-spectrum antitumor activity . Because DHA has been shown to induce immunogenic cell death (ICD) [2-3], we tested whether DHA-dFdC can induce ICD.
Methods: The ability of DHA-dFdC to induce ICD was investigated in CT26 mouse colorectal cancer cells, an established cell line for identifying ICD inducers [4-6], as well as Panc-02 mouse pancreatic cells. Cells were treated with various concentrations of DHA-dFdC and the three surrogate markers of ICD (i.e. calreticulin (CRT) surface translocation, ATP release, and HMGB1 release) were measured. Flow cytometry and confocal microscopy were used to evaluate CRT translocation, a luminescence assay was utilized to measure ATP release, and HMGB1 release was detected using ELISA. To confirm DHA-dFdC’s ability to induce ICD in vivo, CT26 cells treated with DHA-dFdC or oxaliplatin (as a positive control) were implanted subcutaneously in BALB/c mice. One week later, mice were challenged with live CT26 cells and the percent of tumor-free mice was recorded.
Results: In CT26 cells treated with DHA-dFdC, flow cytometry confirmed CRT translocation. Detection of extracellular ATP using a luminescence assay showed ATP release in a dose-response manner following treatment of CT26 cells with various concentrations of DHA-dFdC. Treatment of CT26 cells with DHA-dFdC yielded increased HMGB1 release at 200 µM. Furthermore, pre-treatment of CT26 cells with DHA-dFdC prevented or delayed the development of challenge tumors, similarly to the oxaliplatin positive control (Fig. 1). In Panc-02 cells treated with DHA-dFdC, CRT translocation was also confirmed using flow cytometry and confocal microscopy. Finally, DHA-dFdC induced the release of both extracellular ATP and HMGB1 in Panc-02 cells (Figure 2).
Conclusion: DHA-dFdC induced the surrogate biomarkers of ICD in tumor cells in culture. In addition, immunization of mice with CT26 tumor cells pre-treated with DHA-dFdC prevented or delayed the growth of the same challenge tumor, demonstrating that DHA-dFdC is an ICD inducer.
 Naguib Y, Lansakara-P. D, Lashinger L, Rodriguez B, Valdes S, Niu M et al. Synthesis, Characterization, and In Vitro and In Vivo Evaluations of 4-(N)-Docosahexaenoyl 2′, 2′-Difluorodeoxycytidine with Potent and Broad-Spectrum Antitumor Activity. Neoplasia. 2016;18(1):33-48.
 D’Eliseo D, Di Renzo L, Santoni A, Velotti F. Docosahexaenoic acid (DHA) promotes immunogenic apoptosis in human multiple myeloma cells, induces autophagy and inhibits STAT3 in both tumor and dendritic cells. Genes & Cancer. 2017;8(1-2):426-437.
 Molinari R, D’Eliseo D, Manzi L, Zolla L, Velotti F, Merendino N. The n3-polyunsaturated fatty acid docosahexaenoic acid induces immunogenic cell death in human cancer cell lines via pre-apoptotic calreticulin exposure. Cancer Immunology, Immunotherapy. 2011;60(10):1503-1507.
 Tesniere A, Schlemmer F, Boige V, Kepp O, Martins I, Ghiringhelli F et al. Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene. 2009;29(4):482-491.
 Hossain D, Javaid S, Cai M, Zhang C, Sawant A, Hinton M et al. Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression. Journal of Clinical Investigation. 2018;128(2):644-654.
 Casares N, Pequignot M, Tesniere A, Ghiringhelli F, Roux S, Chaput N et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. The Journal of Experimental Medicine. 2005;202(12):1691-1701.