**1. Introduction**

Photodynamic therapy (PDT) is generally based on three harmless components: molecular oxygen, photosensitizer, and light [1]. Once combined, they yield a robust production of reactive oxygen species (ROS), which can be lethal to the target cell [2–4]. As a cancer treatment modality, PDT can directly kill tumor cells due to its photocytotoxicity, cause infarction of the cancerous tissue by its effects on the tumor microvasculature and activate the immune system against tumor antigens [5]. The activation of the immune system by PDT has been the subject of intense research in recent years. In a murine experimental model of ectopic 4T1 mammary adenocarcinoma, PDT reduced the incidence of metastatic foci in the lungs, even when applied to the primary tumor [6]. Importantly, different parameters of PDT can affect its ability to induce antitumor responses, such as the type [5]

**Citation:** Rodrigues, M.C.; de Sousa Júnior, W.T.; Mundim, T.; Vale, C.L.C.; de Oliveira, J.V.; Ganassin, R.; Pacheco, T.J.A.; Vasconcelos Morais, J.A.; Longo, J.P.F.; Azevedo, R.B.; et al. Induction of Immunogenic Cell Death by Photodynamic Therapy Mediated by Aluminum-Phthalocyanine in Nanoemulsion. *Pharmaceutics* **2022**, *14*, 196. http://doi.org/10.3390/ pharmaceutics14010196

Academic Editor: Maria Nowakowska

Received: 3 December 2021 Accepted: 12 January 2022 Published: 14 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and concentration of photosensitizer, as well as the irradiation regimen [7], a fact that has to be taken into account in the design of PDT-based immunotherapy.

The cell death mechanism triggered by PDT seems to play a key role on its immune effects. For instance, PDT mediated by the photosensitizer aluminum-phthalocyanine (AlPc) can induce both necrosis and apoptosis in murine melanoma B16F10 cells [7]. In this PDT setting, necrosis predominates at higher concentrations of AlPc, while apoptosis is the main cell death mechanism elicited at lower concentrations of this photosensitizer [7]. In the context of antitumor immune responses induced by PDT, the induction of immunogenic cell death (ICD) in target cells is to be cited [8]. According to Garg et al. [5], ICD is an apoptosis modality distinguished by the emission of DAMPs, which are potent immune activators. As agonists of various receptors involved in the immune response, DAMPs can attract and activate different immune cells [9]. They are also capable of promoting proinflammatory events, such as the maturation and activation of antigen-presenting cells, such as dendritic cells and T-cell activating macrophages [8,10].

In this study, the immunogenicity of two murine cancer cell lines–colorectal carcinoma (CT26) and mammary adenocarcinoma (4T1) cells–submitted to different PDT protocols mediated by a nanoemulsion containing aluminum-phthalocyanine (PDT-AlPc-NE) was evaluated. AlPc was chosen as the model photosensitizer because of its high singlet oxygen photogeneration yield and for its efficacy against both primary tumors and metastasis of murine cancer cells, such as 4T1 cells [1,6], in in vivo models. However, the possible immune-related, cellular mechanisms behind the efficacy of AlPc have not been studied in those models. The results show that both CT26 and 4T1 cells emitted different DAMP (calreticulin-CRT, heat shock proteins (HSP)-70 and -90, interleukin 1 beta-IL-1B, and high mobility group-box 1 (HMGB1) after specific PDT-AlPc-NE protocols in vitro. In an in vivo vaccination-challenge model, these PDT-treated cells rendered mice more resistant against the development of experimental tumors.
