Phospholipid scramblase 1 contributes to in vitro antitumor efficacy of dendritic cell vaccine based on the exploitation of tumor cells experiencing immunogenic cell death

MONTICO B; LAMBERTI MJ; NIGRO A; DOLCETTI R; MARTORELLI D; MASTORCI K; RAVO M; VENTOLA G; STEFFAN A; STELLATO C; CASOLARO V; DAL COL J

Congreso; XIII SIICA National Congress; 2022

Whole tumor cell lysates (TCLs) obtained from cancer cells previously killed by treatments able to promote immunogenic cell death (ICD) can be efficiently used as source of tumor-associated antigens for the development of highly efficient dendritic cell (DC)-based vaccines. Phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)-inducible protein with a pro-apoptotic activity in mantle cell lymphoma (MCL) cells treated with the 9-cis-retinoic acid (RA)/ IFN-α combination or doxorubicin, two ICD inducers. Previously we demonstrated that cytotoxic T cells generated with autologous DCs pulsed with RA/ IFNα-treated TCLs more efficiently recognized and specifically lysed MCL cells or targets loaded with several HLA-A*0201 cyclin D1 epitopes. Herein, PLSCR1 contribution in influencing immunogenic features of dying cancer cells and in enhancing RA/IFNα-TCLs DC-based vaccine efficiency was investigated. Methods: PLSCR1 expression was evaluated in different MCL cell lines following ICD induction and different commercial kinase inhibitors were used to identify the signaling pathways involved in its upregulation. Mino and Z-138 cell lines ectopically expressing PLSCR1 were generated to investigate whether this protein affects ICD features. Further, Mino cells ectopically expressing PLSCR1 (Mino–PLSCR1+) were used to obtain whole TCLs for DC loading. Additionally, PLSCR1 was knocked down by short hairpin RNA (Mino-shPLSCR1). The DCs loaded with Mino-PLSCR1+-TCLs or Mino-shPLSCR1-TCLs were employed for generation of tumor- and antigen-specific cytotoxic T lymphocytes. Finally, the transcriptomic profile of Mino-PLSCR1+ cells was defined by next generation sequencing technology. Results: Two different ICD inducers, such as the RA/IFN-α combination and doxorubicin, induced PLSCR1 expression through STAT1 activation. PLSCR1 upregulation favored the proapoptotic effects of RA/IFN-α treatment without influencing any damage-associated molecular patterns (DAMPs) characterizing ICD, such as the exposure of calreticulin and heat shock protein 70 on cell surface or HMGB1 secretion. However, DCs loaded with TCLs obtained from Mino cells ectopically expressing PLSCR1 elicited in vitro higher T cell-mediated antitumor responses compared to DCs loaded with TCLs derived from Mino cells infected with empty vector or the parental cell line. Conversely, PLSCR1 knock-down inhibited the stimulating activity of DCs loaded with RA/IFN-α-treated TCLs to elicit cyclin D1 antigenspecific cytotoxic T lymphocytes. Transcriptomic analysis of MCL cell lines overexpressing PLSCR1 versus those infected with empty vector and subsequent Gene Set Enrichment Analysis highlighted two pathways significantly modulated and likely involved in the immune system response: “Phagocytosis recognition” (activated) and “Negative regulation of innate immune response” (inhibited). Conclusions: Our results indicate that PLSCR1, while not implicated in the control of conventional ICD hallmarks, was clearly involved in DC-based vaccine efficiency, suggesting a potential contribution in the control of pathways associated to DC activation, possibly including those involved in antigen uptake, processing, and presentation.