Aerolysin Cytotoxicity Can Serve as a Surrogate Marker for Immune Sensitivity of Tumor Cells to Granule-Exocytosis Pathway in Vitro.

Background: Aerolysin is a pore-forming toxin produced by Aeromonas hydrophila and interacts with target mammalian cells through binding to glycosylphosphatidyl inositol (GPI)-anchored proteins, subsequently forming a pore in the plasma membrane. Both osmotic lysis of the cell secondary to fluid influx thru pores and a rapid increase in intracellular calcium, which may have been the signal for apoptosis, when a small number of channels were opened appear to be operational in aerolysin cytotoxicity. Perforin (PFP), a prototype of pore-forming proteins is closely linked to granzyme B effect and the central element of natural killer (NK) and T cell mediated cytotoxicity, and induces apoptosis of target cells through granule-exocytosis pathway. Since perforin/granzyme B pathway is pivotal to immune mediated elimination and due to similarities between PFP and aerolysin action, we investigated the relationship between aerolysin cytotoxicity and lymphokine activated killer (LAK) cell-mediated elimination of human tumor cells.

Material and Methods: We studied the effect of active aerolysin (2x10⁻¹⁰M) against eight tumor cell lines and five acute myeloid leukemia patient samples. Aerolysin cytotoxicity was assessed at eight different time points: first at ½ to 4 hours of incubation consistent with the known optimal cell kill mediated by PFP in NK cytotoxicity, then lastly at 18 hours to be able see overall effect. Cytotoxicity was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6 ₍₃₎ /PI staining and further quantification of fluorosphere-adjusted events. Tumor cell type selection was based on their sensitivity against LAK cell-mediated cytotoxicity. Acute myeloid leukemia cells K562, U937, CMK, Meg-01, HL-60 and resistant central nervous system tumor cells (HTB-11, HTB-12 and HTB-14) were included in this study. LAK cell-mediated cytotoxicity against tumor cell lines and patient samples was measured by flow cytometric cell mediated cytotoxicity assay. Drug cytotoxicity was also evaluated in all cell lines and patient samples.

Results: In timing studies, aerolysin treatment reached peak cytotoxicity around 2 hours of incubation. U937 was the most sensitive cell type to both LAK cells and aerolysin. Some cells were very resistant to LAK killing and aerolysin at the concentration used. There was significant correlation between aerolysin cytotoxicity and LAK cell-mediated kill at 2 hours in cell lines and patient samples (r = 0.99; p < 0.001, r = 0.85; p = 0.035, respectively). There was also a statistically significant correlation between optimal dose aerolysin killing at 18 hours of incubation and LAK cell-mediated cytotoxicity (r = 0.94; p = 0.04). However, there was not any correlation between drug cytotoxicity and LAK cell-mediated cytotoxicity or aerolysin-induced cell kill.

Conclusion: Aerolysin sensitivity may be used as a surrogate marker for the assessment of in vitro LAK cell-mediated tumor cell kill through granule/exocytosis pathway.