Investigation of the features of immunogenic cell death caused by photodynamic exposure using a photosensitizer from the tetra(aryl)tetracyanoforfirazines group with 9-phenanthrenyl as a side substituent
Authors: Sleptsova E. E. , Redkin T. S. , Saviuk M. O. , Kondakova E. V. , Vedunova M. V. , Turubanova V. D. , Krysko D. V. 
Published: May 2024

The concept of immunogenic cell death involves the death of tumor cells, leading to the activation of an adaptive immune response in vivo. Such death relies on two significant components: antigenicity and adjuvance of dying cells. The emission of DAMPs achieves adjuvance, which is recognized by antigen-presenting dendritic cell (DC) receptors, resulting in phagocytosis and DC maturation. These cells present antigens of dead cells on their surface to the T-cell population. Antigenicity provides an opportunity to develop adaptive immunity through vaccination with decaying cells targeting a particular tumor pattern (antigen).
Currently, photodynamic therapy (PDT) is recognized as an efficient inducer of immunogenic cell death. In this study, we examined the effectiveness of photodynamic therapy (PDT) using tetracyanotetra(aryl)porphyrazine with a 9-phenanthrenyl group on the periphery of the porphyrazine macrocycle (pz I) as a photosensitizer for inducing immunogenic cell death in tumor cells. The study was conducted on two cell lines: mouse fibrosarcoma MCA205 and mouse glioma GL261.
For photoinduction, cells were loaded with a photosensitizer for four hours. The medium was then replaced with full medium, and the cells were irradiated with a 20 J/cm light dose using an LED light source with an excitation wavelength of 615–635 nm. In all subsequent experiments, cells incubated for 24 hours after photoinduction were used.
A photosensitizer concentration corresponding to 85–90% of dead and dying cells 24 hours after photoinduction was chosen for both cell lines, as it is considered the standard for immunogenic cell death.
The study analyzed the levels of ATP and HMGB1 that were released into the extracellular medium to confirm adjuvanticity. After photodynamic exposure, the level of ATP in the supernatant 24 hours later was significantly higher than the baseline values in the control group prior to PDT for both cell lines. Similar findings were observed for HMGB1 release.
The study aimed to investigate the potential of PDT-killed cells in inducing a persistent immune response. Dying cells of fibrosarcoma MCA205 or GL261 glioma underwent photoinduction using pz I and were used to immunize C57BL/6J mice once or twice a week. After seven days from the last vaccination, viable tumor cells were subcutaneously injected into the opposite side of the mice for observation.
In the MCA205 fibrosarcoma tumor model, 90% of the animals were tumor-free on day 25 of the experiment. In the control group PBS (which comprised mice that received saline solution as immunization), all of the laboratory animals had a tumor by day 12 of observation, and died by day 16. Additionally, on day 16 of the experiment, the volume of tumors in the control group was double the volume of tumors on day 25 of the experiment in the pz I group.
When experimenting with immunization using dying GL261 glioma cells, the pz I group showed an absence of tumor focus in 90% of laboratory animals by day 30. Furthermore, the tumor volume in the group that was immunized with PDT-killed cells was 10 times less compared to the tumor volume of the control group PBS.
Immunization of the Nude strain of bestimus condyles was conducted to evaluate the contribution of adaptive immunity to the manifestation of the antitumor effects of dying cells induced through photodynamic therapy (PDT). Tumor foci appeared and developed similarly in both the experimental and control groups, indicating no significant impact from immunization. Thus, the study demonstrated the critical importance of the T-cell connection in eliciting an effective anti-tumor response. The evidence indicated that if T-cell populations cannot participate in adaptive immune responses, even when immunity is stimulated, a pathological process can develop. Vaccination using photoinduced MCA205 fibrosarcoma cells in immunodeficient Nude mice verified the noteworthy role of the adaptive immune system in executing the antitumor response.