Abstract
BackgroundCancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. IMMUNEPOTENT CRP (ICRP) acts as an immunomodulator and can be cytotoxic to cancer cells. Thus, we evaluated if ICRP induces ICD in BC cells.MethodsImmunogenicity of ICRP-induced cell death was evaluated in vitro, analysing the principal biochemical characteristics of ICD in MCF-7, MDA-MB-231 and 4T1 cells. Ex vivo, we assessed the ability of killed cancer cells (KCC) obtained from ICRP-treated 4T1 cells (ICRP-KCC) to induce DC maturation, T-cell priming and T-cell-mediated cancer cytotoxicity. In vivo, we evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice.ResultsICRP induced caspase-independent, ROS-dependent cell death, autophagosome formation, P-eIF2α, chaperone protein exposure, CD47 loss, ATP and HMBG1 release in BC cells. Additionally, ICRP-KCC promoted DC maturation, which triggered T-cell priming and cancer cytotoxicity. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes.ConclusionsICRP induces ICD in BC cells, leading to long-term antitumour memory.
Highlights
Cancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence
CC50 by IMMUNEPOTENT CRP (ICRP) was caused at 1.25 U/mL in MCF-7 and MDA-MB-231 cells, and 0.15 U/mL in 4T1 cells, whereas CC80 was induced at 1.5 U/mL in human breast cancer cells, and 0.2 U/mL in 4T1 cells, CC100 was reached at 2 U/mL in MCF-7 and MDA-MB-231, and 0.5 U/mL in 4T1 cells, where we observed more than 90% of cell death (Fig. 1a)
Loss of mitochondrial membrane potential was induced in 55–65% of MCF-7, MDA-MB231 and 4T1 cells treated with ICRP CC50 for 24 h (Fig. 1b), this treatment generated an increase of reactive oxygen species (ROS) levels in 45–55% of MCF7, MDA-MB-231 and 4T1 cells (Fig. 1c)
Summary
Cancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. We evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes. IMMUNEPOTENT CRP (ICRP), a bovine dialysable leukocyte extract (DLE) obtained from disrupted spleen, is cytotoxic to several cancer cell lines,[8,9,10,11] without affecting the viability of noncancer cells.[11] ICRP induces ICD in the murine melanoma model B16F10,12 whereas in HeLa and MCF-7 cells, ICRP-mediated cell death involves CRT exposure, ATP and HMGB1 release, which are the principal damage-associated molecular patters (DAMPs) involved in ICD. ICRP has been reported to induce reactive oxygen species (ROS)-dependent autophagosome formation in HeLa and MCF-7 cells.[13]
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