Gold standard assessment of immunogenic cell death induced by photodynamic therapy: From in vitro to tumor mouse models and anti-cancer vaccination strategies.

The discovery of the concept of immunogenic cell death (ICD) is a cornerstone in the development of novel anti-cancer immunotherapeutic approaches. Induction of the ICD pathway by specific anti-cancer therapeutic regimens can eliminate cancer cells by directly killing them during therapy and by activation of strong and specific anti-cancer immunity, leading to a long-lasting immunological memory that prevents cancer recurrence. ICD encompasses different forms of regulated cell death and can be triggered by many anti-cancer treatment modalities, including photodynamic therapy (PDT). PDT is a multistep procedure involving the accumulation of a light-sensitive dye known as a photosensitizer (PS) in tumor cells, followed by its activation by irradiation with a light of an appropriate wavelength. In the presence of molecular oxygen, the irradiated PS leads to the generation of cytotoxic reactive oxygen species, which can lead to ICD induction in the cancer cells. Here, we first describe in vitro methods to help optimize the PDT procedure for a specific PS. We also provide a collection of protocols and techniques for assessing ICD in vitro, including analysis of the emission of damage associated molecular patterns (DAMPs), efferocytosis, and the maturation and activation state of antigen presenting cells. Next, we describe in detail protocols for diverse tumor mouse models for assessing and characterizing ICD in vivo, such as murine tumor vaccination models. Finally, as an immunotherapeutic vaccine, we suggest using either PDT-induced dead cancer cells, preferably undergoing ICD, or dendritic cells loaded with lysates of PDT-induced cancer cells in a syngeneic orthotopic glioma model. Overall, this methodological article provides a quantitative, comprehensive set of validated tools that can be successfully used, with some adaptations, to identify, optimize and validate novel PSs in vitro and in vivo for the efficient induction of ICD during photodynamic treatment.

CX3CL1 release during immunogenic apoptosis is associated with enhanced anti-tumour immunity.

Introduction: Immunogenic cell death (ICD) has emerged as a novel option for cancer immunotherapy. The key determinants of ICD encompass antigenicity (the presence of antigens) and adjuvanticity, which involves the release of damage-associated molecular patterns (DAMPs) and various cytokines and chemokines. CX3CL1, also known as neurotactin or fractalkine, is a chemokine involved in cellular signalling and immune cell interactions. CX3CL1 has been denoted as a "find me" signal that stimulates chemotaxis of immune cells towards dying cells, facilitating efferocytosis and antigen presentation. However, in the context of ICD, it is uncertain whether CX3CL1 is an important mediator of the effects of ICD. Methods: In this study, we investigated the intricate role of CX3CL1 in immunogenic apoptosis induced by mitoxantrone (MTX) in cancer cells. The Luminex xMAP technology was used to quantify murine cytokines, chemokines and growth factors to identify pivotal regulatory cytokines released by murine fibrosarcoma MCA205 and melanoma B16-F10 cells undergoing ICD. Moreover, a murine tumour prophylactic vaccination model was employed to analyse the effect of CX3CL1 on the activation of an adaptive immune response against MCA205 cells undergoing ICD. Furthermore, thorough analysis of the TCGA-SKCM public dataset from 98 melanoma patients revealed the role of CX3CL1 and its receptor CX3CR1 in melanoma patients. Results: Our findings demonstrate enhanced CX3CL1 release from apoptotic MCA205 and B16-F10 cells (regardless of the cell type) but not if they are undergoing ferroptosis or accidental necrosis. Moreover, the addition of recombinant CX3CL1 to non-immunogenic doses of MTX-treated, apoptotically dying cancer cells in the murine prophylactic tumour vaccination model induced a robust immunogenic response, effectively increasing the survival of the mice. Furthermore, analysis of melanoma patient data revealed enhanced survival rates in individuals exhibiting elevated levels of CD8+ T cells expressing CX3CR1. Conclusion: These data collectively underscore the importance of the release of CX3CL1 in eliciting an immunogenic response against dying cancer cells and suggest that CX3CL1 may serve as a key switch in conferring immunogenicity to apoptosis.

Glioma: bridging the tumor microenvironment, patient immune profiles and novel personalized immunotherapy.

Glioma is the most common primary brain tumor, characterized by a consistently high patient mortality rate and a dismal prognosis affecting both survival and quality of life. Substantial evidence underscores the vital role of the immune system in eradicating tumors effectively and preventing metastasis, underscoring the importance of cancer immunotherapy which could potentially address the challenges in glioma therapy. Although glioma immunotherapies have shown promise in preclinical and early-phase clinical trials, they face specific limitations and challenges that have hindered their success in further phase III trials. Resistance to therapy has been a major challenge across many experimental approaches, and as of now, no immunotherapies have been approved. In addition, there are several other limitations facing glioma immunotherapy in clinical trials, such as high intra- and inter-tumoral heterogeneity, an inherently immunosuppressive microenvironment, the unique tissue-specific interactions between the central nervous system and the peripheral immune system, the existence of the blood-brain barrier, which is a physical barrier to drug delivery, and the immunosuppressive effects of standard therapy. Therefore, in this review, we delve into several challenges that need to be addressed to achieve boosted immunotherapy against gliomas. First, we discuss the hurdles posed by the glioma microenvironment, particularly its primary cellular inhabitants, in particular tumor-associated microglia and macrophages (TAMs), and myeloid cells, which represent a significant barrier to effective immunotherapy. Here we emphasize the impact of inducing immunogenic cell death (ICD) on the migration of Th17 cells into the tumor microenvironment, converting it into an immunologically "hot" environment and enhancing the effectiveness of ongoing immunotherapy. Next, we address the challenge associated with the accurate identification and characterization of the primary immune profiles of gliomas, and their implications for patient prognosis, which can facilitate the selection of personalized treatment regimens and predict the patient's response to immunotherapy. Finally, we explore a prospective approach to developing highly personalized vaccination strategies against gliomas, based on the search for patient-specific neoantigens. All the pertinent challenges discussed in this review will serve as a compass for future developments in immunotherapeutic strategies against gliomas, paving the way for upcoming preclinical and clinical research endeavors.