Light-Regulated Cancer Immunotherapy Using Individually Encapsulated Synthetic Circuit–Engineered Cells

Background

Cell therapy has been widely explored in regenerative medicine and cancer immunotherapy [1]. By administering living cells with defined therapeutic functions, such as stem cells or immune cells, this approach aims to repair damaged tissues, modulate immune responses, or eliminate malignant cells [2–5]. Among these modalities, chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable clinical success in hematologic malignancies and has provided an effective option for patients with relapsed or refractory disease [6–7]. Likewise, mesenchymal stem cells (MSCs), owing to their immunomodulatory, tissue-reparative, and homing properties, have shown promise in clinical studies of autoimmune diseases, myocardial infarction, and graft-versus-host disease (GVHD), with more than one thousand registered trials worldwide [8–10].

Despite these advances, poor post-transplant survival and limited homing efficiency remain major barriers to clinical translation [11]. Following intravenous infusion or local delivery, transplanted cells are exposed to hypoxia, ischemia, and inflammatory stress, resulting in substantial cell loss before reaching the target tissue. For example, more than 90% of intravenously administered MSCs are trapped in the lungs within 24 h, and only a small fraction successfully home to target organs [12]. For CAR-T cells, the physical barriers of solid tumors, aberrant vasculature, and immunosuppressive tumor microenvironments likewise restrict infiltration and persistence [13–14]. In addition, allogeneic cell products, including off-the-shelf CAR-T cells and donor-derived MSCs, may be rapidly cleared by host immune responses, thereby limiting therapeutic efficacy [15].

To overcome these limitations, researchers have been actively exploring novel strategies for spatiotemporally precise regulation of therapeutic cell functions [16]. In recent years, optogenetic-based gene switch technologies, particularly the red/far-red light reversible regulatory system, have provided a new solution for cancer immunotherapy. Compared with blue light systems, red light exhibits deeper tissue penetration (up to several centimeters) and lower photocytotoxicity, making it more suitable for in vivo applications. A typical red light–controlled gene switch utilizes phytochromes (e.g., Arabidopsis PhyB) and their interacting factors (PIFs). Under red light irradiation (660 nm), phytochrome binds to PIF, thereby initiating downstream gene transcription; upon far-red light irradiation (730 nm), they dissociate, leading to rapid termination of gene expression [17]. This red/far-red light bidirectional switch features high spatiotemporal resolution, reversibility, and fast response kinetics. It has been successfully applied to regulate the expression of therapeutic cytokines (e.g., IL-2, IFN-γ) in CAR-T cells, the on-demand release of immune checkpoint inhibitors, and the membrane localization of chimeric antigen receptors. Compared with conventional constitutive expression or chemically inducible systems, the red light–controlled gene switch enables the stringent restriction of therapeutic protein expression to the tumor site and within a defined time window, thereby substantially reducing systemic immune-related toxicities. When integrated into the synthetic gene circuits of engineered cells, such optogenetic switches allow remote and reversible control over immune response intensity, opening a new avenue for precision cancer immunotherapy.

Meanwhile, cell surface engineering has also attracted much attention as an important strategy for improving the survival and function of transplanted cells [18–19]. As the interface between cells and their environment, the cell surface plays a central role in adhesion, signaling, immune recognition, and homing. Surface engineering enables the introduction of additional functions without altering the cell genome, thereby allowing precise control over cellular behavior [20]. Existing approaches include physical encapsulation with biocompatible coatings, chemical conjugation of functional molecules, and metabolic incorporation of non-native groups into membrane components [21–23]. Protective coatings based on hydrogels or polyelectrolyte membranes, as well as membrane-anchored immunomodulatory molecules, can reduce immune recognition and prolong the in vivo persistence of allogeneic cells. Such coatings may also act as local delivery platforms, creating a supportive pericellular microenvironment that enhances cell survival and functional stability [24–25].

In this protocol, we developed a cell surface encapsulation strategy based on the HRP-pH-low insertion peptide (pHLIP). HRP-pHLIP was inserted into the cell membrane, and in the presence of H₂O₂, horse radish peroxidase (HRP) catalyzed the crosslinking of hyaluronic acid–dopamine (HA-DA), enabling in situ encapsulation of individual cells. This approach integrates targeted membrane insertion with enzyme-mediated crosslinking to generate a mild, controllable protective coating, with the goal of improving cell survival under hostile post-transplantation conditions.