Orchestrated Endoplasmic Reticulum-directed Immunogenic Cell Death Enabled by Indole-derived Covalent Donor-acceptor Dyad via Triplet State Hydrogen Atom Transfer

Photoimmunotherapy (PIT) integrates photodynamic therapy (PDT) to activate systemic antitumor immunity against immunogenic cold tumors and their recurrence. Although ER-targeted photosensitizers offer an optimal solution by direct immune activation, their clinical translation is constrained by tumor hypoxia, poor accumulation, high diffusivity, dependence on a heavy atom for Type-I PDT, and an elusive mechanism underlying Type-I photosensitization. This work presents a structurally minimalistic, heavy-atom-free indole-based covalent photosensitizer (C2) that achieves Type I photosensitization via an unprecedented triplet-state hydrogen atom transfer mechanism. Remarkably, C2 enables N–H homolysis to generate H• and indole radicals under ultra-low dose of (3.47 mW/cm2) green light. Intriguingly, C2 imparts excellent photostability, negligible dark cytotoxicity, and is capable of activating adaptive immune response towards dying tumor cells. Structurally, C2 integrates an indole donor with nitrile acceptors that also serve as electrophilic handles for covalent protein modification. A bis-trifluoromethylphenyl moiety directs selective ER localization, enabling localized ROS generation and sustained immune activation through targeted covalency. Under dark conditions, C2 was found to elicit autophagy as a cell survival mechanism to counteract the unfolded protein response (UPR); however, upon light exposure, C2 undergoes photosensitization to induce severe oxidative damage via generated singlet oxygen (1O2) and superoxide radicals (O2•-) within the ER, which leads to the massive releases of damage-associated molecular patterns (DAMPs) as a danger signal to ameliorate immunogenic cell death (ICD) and apoptosis. The efficacy of PIT was further verified using an ultra-large (Diameter ˃1500 µm) hypoxic 3D-spheroid model to mimic solid tumor conditions.