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. 2025 Nov 4;147(48):44131–44140. doi: 10.1021/jacs.5c13053

Optogenetic Cancer Therapy Using the Light-Driven Outward Proton Pump Rhodopsin Archaerhodopsin‑3 (AR3)

Shin Nakao †, Keiichi Kojima †,‡,*, Keita Sato †,‡, Naoya Kemmotsu †,§, Hideyo Ohuchi †,‡, Yosuke Togashi †,‡,∥,⊥,#, Yuki Sudo †,‡,*
PMCID: PMC12723686  PMID: 41186408

Abstract

Medicines used for cancer treatment often cause serious side effects by damaging normal cells due to nonspecific diffusion. To address this issue, we previously developed an optical method to induce apoptotic cell death via intracellular pH alkalinization using the outward proton pump rhodopsin, Archaerhodopsin-3 (AR3) in various noncancer model cells in vitro and in vivo. In this study, we applied this method to cancer cells and tumors to evaluate its potential as an anticancer therapeutic strategy. First, we confirmed that AR3-expressing murine cancer cell lines (MC38, B16F10) showed apoptotic cell death upon green light irradiation, as indicated by increased levels of cell death and apoptosis-related markers. Next, we established stable AR3-expressing MC38 and B16F10 cells by using viral vectors. When these AR3-expressing cells were subcutaneously transplanted into C57BL/6 mice, the resulting tumors initially grew at a rate comparable to that of control tumors lacking AR3 expression or light stimulation. However, upon green light irradiation, AR3-expressing tumors exhibited either a marked reduction in size or significantly suppressed growth, accompanied by the induction of apoptosis signals and decreased proliferation signals. These results demonstrate that AR3-mediated cell death has potent antitumor effects both in vitro and in vivo. This optical method thus holds promise as a novel cancer therapy with potentially reduced side effects.

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Introduction

The homeostasis of living organisms, including humans, is maintained by a balance between cell production and cell death, and the disruption of this balance can lead to various diseases. Among the forms of cell death, apoptosis is a highly conserved cell death mechanism that is observed across multicellular organisms, including humans. This tightly regulated mechanism plays essential roles in development, tissue maintenance, and immune defense. , Dysregulation of the apoptosis signaling pathway is implicated in a wide range of human disorders. Indeed, excessive activation contributes to conditions such as neurodegenerative diseases and AIDS, whereas insufficient induction leads to the accumulation of abnormal cells, as seen in cancer. , In cancer, apoptotic signaling is frequently impaired through various molecular alterations affecting both the intrinsic and extrinsic pathways. , These include overexpression of antiapoptotic Bcl-2 family proteins (e.g., Bcl-2 and Bcl-xL), downregulation or inactivation of pro-apoptotic members such as Bcl-2-Associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), loss-of-function mutations in tumor suppressors like p53, and elevated levels of inhibitors of apoptosis proteins (IAPs) that suppress caspase activation. Collectively, these disruptions enable tumor cells to evade apoptosis, leading to an uncontrolled proliferation. Nevertheless, apoptosis remains a key target in cancer treatment due to its primarily noninflammatory nature. To date, numerous chemotherapeutic agents (e.g., platinum-based drugs, DNA synthesis inhibitors, replication blockers and microtubule-targeting drugs) have been developed to induce apoptosis in malignant cells, − often by reactivating partially retained apoptotic pathways. ,, Therefore, the development of diverse strategies that activate apoptosis through different molecular mechanisms is essential for overcoming the broad variability in apoptotic resistance observed across different cancer types.

Recently, optogenetics has emerged as a powerful approach to optically controlling cellular activities, in which photoreceptive proteins initiate specific cellular processes. , Light stimulation can induce cellular responses in target cells where photoreceptive proteins are ectopically expressed and localized via genetic methods. , Compared to conventional chemical methods, optogenetics offers the distinct advantages of precise and reversible manipulation of biological functions with high spatiotemporal resolution. − This capability has significantly advanced both basic biological research and the development of therapeutic strategies. ,, Among their diverse applications, optogenetic strategies have been employed to regulate apoptosis pathways. For example, pro-apoptotic proteins such as caspase-3, caspase-8, caspase-9 and Bax have been fused with light-sensitive domains derived from flavin-based proteins, including Cryptochrome 2 (CRY2) and the Light-Oxygen-Voltage-sensing domain 2 (LOV2). These engineered constructs enable light-dependent control of apoptotic signaling, leading to the induction of apoptosis. − In addition, another photoreceptive protein, microbial rhodopsin, is also used for the induction of apoptosis. − Microbial rhodopsin is a family of photoreceptive seven-transmembrane proteins in which chromophore retinal is bound to conserved lysine residue at seventh helix of the apoprotein (Figure S1A). , They are widely utilized in optogenetics for their light-induced molecular functions, including ion channeling, ion pumping, and enzymatic activities. In our previous study, we established a novel optogenetic method to induce apoptotic cell death via intracellular pH alkalinization using the robust outward proton pump rhodopsin Archaerhodopsin-3 (AR3). We demonstrated that light activation of AR3 triggered pH increases from 7.4 to 8.5 in 5 min and effectively induced apoptosis in multiple human cell lines, including HeLa and SH-SY5Y, in vitro. Furthermore, light activation of AR3 in amphid sensory neurons induced a significant depression of chemotaxis in the nematode Caenorhabditis elegans, strongly suggesting light-dependent induction of apoptosis in amphid neurons in vivo. Thus, this light-induced apoptosis method indicated that intracellular alkalinization can serve as a key trigger for apoptosis signaling cascades and showed high applicability both in vitro and in vivo. These findings raise an open question of whether this approach could be applied as a therapeutic strategy for cancer (Figure A).

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Light-induced cell death in AR3-transfected MC38 and B16F10 cells in vitro. (A) Schematic illustration of AR3-dependent light-induced apoptosis. Upon green light illumination, AR3 pumps protons out of the cell, leading to intracellular alkalinization, which induces mitochondrial-mediated signaling in cancer cells. (B, D) Detection of propidium iodide (PI) fluorescence signals in mock and AR3-expressing MC38 (B) and B16F10 cells (D) before and after irradiation. Merged fluorescence images, including PI, EYFP and Hoechst, are shown. Scale bars represent 50 μm. (C, E) Fraction of PI-positive cells in mock and AR3-expressing MC38 (C) and B16F10 (E) cells. Data are presented as the means ± SEM from three biological replicates, each analyzing >103 cells.

Cancer-specific cell lines (i.e., cancer model cells), which are derived from tumors and replicate key phenotypic features such as gene expression profiles and signal transduction pathways of cancer tissues, often exhibit heterogeneity in their susceptibility to apoptosis and display different responses to anticancer drugs compared to commonly used cell lines, such as HeLa, HEK293 and NIH-3T3, in vitro. , Moreover, cancer model cells show markedly different responses to anticancer therapies depending on the host immune environment, as demonstrated by immunocompetent syngeneic mouse models versus immunodeficient xenograft mouse models. , These findings highlight that true therapeutic efficacy cannot be accurately assessed without an intact immune system. Therefore, it is essential to evaluate the anticancer effects of AR3-mediated light-induced alkalinization in cancer cells both in vitro and in vivo, particularly within syngenetic hosts that preserve tumor-immune interactions. This represents a crucial first step toward assessing the therapeutic potential of this optogenetic approach. In this study, we applied our AR3-based optogenetic strategy to murine cancer cell lines and demonstrated its high efficacy in inducing apoptosis and antitumor effects both in vitro and in vivo using immunocompetent syngeneic mouse models. Based on these findings, we discuss the therapeutic potential of this optogenetic approach for cancer treatment.

Results

AR3-Dependent Light-Induced Cell Death in Cancer Model Cells

To assess the applicability of our optogenetic strategy to biologically relevant cancer models, we heterologously expressed AR3 in the murine colorectal cancer cell line MC38 and murine melanoma cell line B16F10 using a transient transfection system (Figure S2A) according to the method as reported previously. EYFP fluorescence, fused to the C-terminus of AR3, was predominantly detected at the periphery of transfected cells (Figure S2B), indicating that AR3 was mainly localized to the plasma membrane. To evaluate light-induced cell death, both AR3-transfected and untransfected (mock) cells were irradiated with green light (545 ± 10 nm, 7 mW/mm2) (Figures S1B and S2C) and stained with propidium iodide (PI), a fluorescent dye that marks cells with compromised membrane integrity. Following light irradiation, PI-positive signals increased in a time-dependent manner in AR3-transfected MC38 and B16F10 cells, whereas no significant PI signals were observed in mock cells (Figure B-E). This time-dependent increase in PI-positive cells suggests progressive light-induced cell death mediated by AR3 in both cell lines. Notably, the proportion of PI-positive cells reached approximately 50% in MC38 and 90% in B16F10 cells, levels comparable to or even exceeding those observed in AR3-transfected HeLa cells. These results demonstrate that AR3 activation effectively induces cell death, despite differences in apoptotic susceptibility across various cancer and noncancer model cell lines. It should be noted that light-induced cell death did not reach 100%, likely owing to insufficient AR3 expression in the transfected cells, indicating that illumination alone is unlikely to completely eradicate a tumor.

In our previous study, we demonstrated that light activation of AR3 induces mitochondrial-mediated apoptosis in HeLa cells. To determine whether a similar apoptotic mechanism occurs in cancer model cells, we investigated mitochondrial membrane potential (ΔΨm) collapse in AR3-transfected MC38 and B16F10 cells using a tetramethylrhodamine ethyl ester (TMRE) assay. In this assay, the fluorescence intensity decreases upon the loss of ΔΨm, a hallmark of early stage mitochondrial apoptosis. As cell death appeared to plateau after 120 min of irradiation (Figure ), we assessed TMRE fluorescence before and after 120 min of irradiation. After 120 min of green light irradiation (545 ± 10  nm, 7 mW/mm2), AR3-transfected MC38 and B16F10 cells exhibited a remarkably reduction in TMRE fluorescence intensity, indicating ΔΨm collapse (Figure A, B). In contrast, mock cells showed no significant change. The proportion of TMRE-negative cells after light irradiation was estimated to be 44% in MC38 and 62% in B16F10 cells (Figure B), consistent with the cell death rates observed after 120 min of irradiation (55 and 95% in MC38 and B16F10 cells, respectively; Figure ). These findings strongly suggest that light-induced cell death in AR3-transfected cells is associated with early mitochondrial-mediated apoptotic signaling. To further confirm the activation of apoptosis, we evaluated caspase-3 activation and DNA fragmentation, hallmarks of the late phase of mitochondrial-mediated apoptosis, using CellEvent and TUNEL assays, respectively. Both assays demonstrated increased fluorescence in AR3-transfected MC38 and B16F10 cells following 120 min of irradiation, while mock cells remained negative (Figures C–F). The proportions of CellEvent- and TUNEL-positive cells were estimated to be 51 and 50% in MC38 cells and 63 and 73% in B16F10 cells, respectively (Figure D and F), consistent with the PI-positive cell death rates (Figure C and E). Furthermore, we assessed the activation of BAX, a key apoptosis regulator that interacts with mitochondria to trigger mitochondrial-mediated signaling. However, immunostaining with the conformation-specific BAX antibody (6A7) revealed no significant increase in fluorescence intensity following light stimulation, suggesting that conformational activation of BAX was not induced under these conditions and that the light-induced cell death may occur through a BAX-independent mechanism (Figure S3). Collectively, these results demonstrate that the AR3-based optogenetic method induces both early and late phases of mitochondrial-mediated apoptosis in cancer model cells in vitro, highlighting its potential as a precise and effective strategy for targeted cancer cell elimination. A recent study demonstrated that AR3-induced cytosolic alkalinization results in increased production of reactive oxygen species (ROS) in both the cytosol and mitochondria. Accordingly, we examined ROS levels in MC38 and B16F10 cells using the ROS probe CellROX. ROS levels were significantly elevated after 30 min of irradiation (Figure S4A), indicating that a similar increase in ROS generation occurred in both MC38 and B16F10 cells. Furthermore, to investigate whether intracellular alkalinization triggers endoplasmic reticulum (ER)-mediated apoptosis by affecting ER function, we monitored intracellular Ca2+ dynamics using the Ca2+ probe Fura-2. An increase in intracellular Ca2+ levels was observed in both AR3-transfected MC38 and B16F10 cells after 5 min of irradiation. These findings suggest that intracellular alkalinization induces ER-mediated apoptosis in addition to mitochondria-mediated apoptosis.

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Apoptosis signaling pathway of AR3-transfected MC38 and B16F10 cells upon irradiation. (A, C, E) TMRE (A), CellEvent (C) and TUNEL (E) assays of MC38 (left panels) and B16F10 (right panels) cells before and after 120 min irradiation. Fluorescence data from mock and AR3-expressing cells are shown in the upper and lower panels, respectively. Merged fluorescence images including EYFP and Hoechst signals are shown. White arrows indicate representative TMRE-negative (A), CellEvent-positive (C), and TUNEL-positive (E) cells. Scale bars represent 50 μm. The cells were irradiated at 545 ± 10 nm (7 mW/mm2). (B, D, F) Fraction of TMRE-negative (B), CellEvent-positive (D), and TUNEL-positive (F) cells in mock and AR3-expressing cells before and after 120 min irradiation. Data are presented as the means ± SEM from three biological replicates, each analyzing >60 cells. Asterisks indicate statistically significant differences between the values (**P < 0.01, ***P < 0.001; Student’s t test).

AR3-Dependent Light-Induced Cell Death in a Living Mouse Tumor Model

To evaluate the applicability of our optogenetic method in vivo, we established stably AR3-expressing MC38 and B16F10 cells using lentiviral or retroviral transduction for subsequent transplantation into mice according to the method as reported previously (Figure S5A). , EYFP fluorescence, fused to the C-terminus of AR3, was predominantly observed at the cell periphery (Figure S5B), suggesting plasma membrane localization. The fluorescence signal remained stable over multiple cell passages (Figure S5C), indicating sustained AR3 expression. To verify whether stably expressed AR3 could trigger cell death upon light stimulation, as seen in transient expression systems (Figure ), we irradiated the stable cell lines with green light for 60 min (Figure S6). PI staining experiments revealed robust cell death following irradiation with estimated cell death rates of 50 and 72% in AR3-expressing MC38 and B16F10 cells, respectively (Figure S6B). These values were comparable to those observed in transiently transfected cells (47 and 72% in MC38 and B16F10 cells, respectively; Figure ). These results indicate that stable AR3 expression retains light-inducible cytotoxicity in vitro.

Next, we subcutaneously transplanted AR3-expressing MC38 and B16F10 cells into mice to generate solid tumors (Figure S7A). EYFP fluorescence was detected in tumor tissues derived from both cell lines (Figure S7B). On day 6 post-transplantation, tumors were irradiated with a green laser (532 ± 1 nm, 7 mW/mm2) for 60 min under general anesthesia (Figures A, S1B). To assess apoptosis induction, cleaved caspase-3 immunohistochemistry was performed on tumor sections collected immediately after light irradiation (i.e., on day 6 post-transplantation). Across all conditions, cleaved caspase-3-positive cells were found in the inner tumor regions distal to the skin surface (Figures B,C and S8A,B), likely reflecting apoptosis induced by hypoxia or nutrient deprivation in the tumor core. In contrast, strong caspase-3 signals were observed at the tumor surface exclusively in AR3-expressing tumors following light irradiation (Figures B,C and S8A,B). To delineate the spatial distribution of apoptotic cells, we quantified the cleaved caspase-3 signal intensity across tissue depth from the body surface. In AR3-expressing tumors, the signal was significantly increased, extending up to approximately 1.2 mm and 1.0 mm from the body surface in MC38 and B16F10 tumors, respectively (Figure D,E). Furthermore, quantitative analysis confirmed a significant increase in cleaved caspase-3-positive cells under these conditions (Figure F,G), indicating that light-activated AR3 selectively induces apoptosis in the surface regions of tumors. These findings suggest that light irradiation effectively induced apoptosis in AR3-expressing tumors, predominantly within the outer layers of the tumor tissue. To examine the effects on tumor cell proliferation, we performed immunohistochemical staining for phosphorylated histone H3 (PHH3), a mitosis marker. In all groups except irradiated AR3-expressing tumors, PHH3-positive cells were consistently detected near the tumor surface (Figures A,B and S9A,B). However, these proliferative signals were nearly absent in irradiated AR3-transduced tumors. Quantification revealed a significant reduction in PHH3-positive cells in the surface regions of AR3-expressing MC38 and B16F10 tumors after light exposure (Figure C,D), suggesting the suppression of mitotic activity. In contrast, neither an increase in apoptosis nor a reduction in proliferation was observed in deeper tumor regions (>1.5 mm from the surface) of irradiated AR3-expressing tumors (Figures S8C and S9C), likely due to limited tissue penetration of green light. Collectively, these findings demonstrate that light activation of AR3 in vivo can induce localized apoptosis and suppress tumor cell proliferation at the illuminated surface of solid tumors, highlighting the spatial specificity and therapeutic potential of this optogenetic cancer treatment strategy.

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Apoptosis induction of AR3-transduced MC38 and B16F10 tumor tissues upon irradiation. (A) Representative image of the in vivo illumination setup used to activate AR3 in subcutaneous tumors. Tumor-bearing C57BL/6J mice were anesthetized and exposed to continuous green light (532 ± 1 nm, 7 mW/mm2) using a laser source positioned 45 cm above the tumor site. (B, C) Representative images of hematoxylin and cleaved caspase-3 staining of MC38 (B) and B16F10 (C) tumor tissues on day 6 after the transplantation. Light irradiation was performed on day 6. Upper panels show low-magnification overviews of the tumor tissues; lower panels show higher-magnification views of the boxed areas in the upper panels. The top of each image corresponds to the skin surface, and the bottom to the deeper tissue region. Scale bars represent 1 mm and 50 μm in the upper and lower panels, respectively. (D, E) Cleaved caspase-3 signal intensity profiles across tissue depth from the epidermal side of the MC38 (D) and B16F10 (E) tumor tissues. Each line represents the mean signal intensity (a.u.) with shaded areas indicating SEM. Asterisks indicate statistically significant differences between mock without irradiation and AR3 with irradiation within the marked depth range (*P < 0.05; Dunnett’s test). (F, G) Fraction of cleaved caspase-3-positive cells of MC38 (F) and B16F10 (G) tumor tissues. Data are presented as the means ± SEM from three biological replicates, each analyzing >264 cells. Asterisks indicate statistically significant differences between the values (***P < 0.001; Dunnett’s test). ″+Irr″ and ″–Irr″ indicate the presence and absence of light irradiation, respectively.

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Proliferative suppression of AR3-transduced MC38 and B16F10 tumor tissues upon irradiation. (A, B) Representative images of hematoxylin and phosphorylated histone H3 (PHH3) staining of MC38 (A) and B16F10 (B) tumor tissues on day 6 after the transplantation. Light irradiation was performed on day 6. Upper panels show low-magnification overviews of the tumor tissues; lower panels show higher-magnification views of the boxed areas in the upper panels. The top of each image corresponds to the skin surface, and the bottom to the deeper tissue region. Scale bars represent 1 mm and 50 μm in the upper and lower panels, respectively. Arrows indicate representative PHH-positive cells. ″+Irr″ and ″–Irr″ indicate the presence and absence of light irradiation, respectively. (C, D) Fraction of PHH3-positive cells of MC38 (C) and B16F10 (D) tumor tissues. Data are presented as the means ± SEM from three biological replicates, each analyzing >328 cells. Asterisks indicate statistically significant differences between the values (*P < 0.05, ***P < 0.001; Dunnett’s test).

Given the potential for off-target effects in optogenetic cancer treatment strategies, we assessed whether green laser irradiation induces apoptosis in surrounding immune cells such as macrophages and dendritic cells, which are closely associated with tumor progression. Macrophages and dendritic cells in MC38 and B16F10 tumor tissues were stained with F4/80 and CD11c antibodies, respectively (Figures and S10). In both mock- and AR3-transduced tumor groups, cleaved caspase-3–positive immune cells were rarely observed in or around the tumors regardless of irradiation. Indeed, the fraction of cleaved caspase-3–negative immune cells was close to 100% and comparable with and without irradiation in both groups (Figures C,D and S10C,D), indicating that green laser illumination does not significantly induce off-target apoptosis of surrounding immune cells. Moreover, the number of immune cells within AR3-transduced tumors rather than in adjacent epidermal tissues was significantly higher with irradiation than without irradiation (Figures E,F and S10E,F). This suggests that following irradiation, immune cells mounted an antitumor response by infiltrating apoptosis-induced tumor cells. These findings indicate that green laser irradiation neither disrupts immune cell homeostasis nor causes phototoxicity, while preserving immune cell infiltration into tumors.

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Distribution of immune cells in MC38 and B16F10 tumor tissues. (A, B) Merged fluorescence images showing macrophages (F4/80–positive), cleaved caspase-3, and DAPI signals in MC38 (A) and B16F10 (B) tumor tissues on day 7 after transplantation. Upper panels show low-magnification overviews of the tumor tissues, while lower panels show higher-magnification views of the boxed areas in the upper panels. White dotted lines indicate the boundary between tumor and epidermal regions (upper and lower sides, respectively). Scale bars represent 100 and 25 μm in the upper and lower panels, respectively. (C, D) Fraction of cleaved caspase-3–negative macrophages in MC38 (C) and B16F10 (D) tumor tissues. Data are presented as mean ± SEM from three biological replicates, each analyzing 5–102 cells. No significant differences were observed between groups (P > 0.05; Dunnett’s test). (E, F) The number of macrophages infiltrating tumor regions in MC38 (E) and B16F10 (F) tumor tissues. Data are presented as mean ± SEM from three biological replicates. Asterisks indicate significant differences between the groups (**P < 0.01, ***P < 0.001; Dunnett’s test).

Antitumor Effect by AR3 upon Light Stimulation In Vivo

As light-dependent apoptotic cell death was observed in AR3-transduced tumor tissues in living mice (Figures and ), we next investigated whether this optogenetic approach could elicit a measurable antitumor effect in vivo. Following subcutaneous transplantation, tumors were irradiated with a green laser for 60 min under general anesthesia on day 6. Tumor growth was subsequently monitored for 13 days (Figure ). The sizes of AR3-transduced MC38 and B16F10 tumors subjected to light irradiation were visibly smaller than those in all control groups (Figure A and D). Quantitative analysis confirmed that tumor volumes in the irradiated AR3-expressing groups were significantly reduced in comparison with all other groups on day 13 (Figure B and E). Importantly, no significant differences in tumor size were observed between groups prior to light treatment (days 4 and 6), indicating that the observed suppression of tumor growth was specifically attributable to AR3 activation. Of note, AR3-transduced MC38 tumors continued to shrink from days 10 to 13, suggesting that while complete regression did not occur, tumor progression was still effectively inhibited by AR3-mediated light stimulation. To evaluate potential systematic toxicity associated with this optogenetic treatment, given that many anticancer drugs induce adverse effects such as significant body weight loss, we monitored the body weights of mice bearing either mock- or AR3-transduced tumors throughout the experimental period. No notable changes in body weight were observed in mice subjected to light activation of AR3 on days 10 and 13 compared to their weights prior to light irradiation (days 0, 4, and 6) (Figure C and F). Quantitative assessment confirmed that body weights remained stable and were not significantly different from those of the control groups. Taken together, these findings demonstrate that light activation of AR3 induces a robust antitumor effect in vivo, characterized by tumor growth suppression in both models and tumor regression, specifically in MC38 tumors, without causing gross systemic toxicity. Importantly, this optogenetic approach did not induce overt systemic toxicity, highlighting its potential as a spatially controllable and minimally invasive cancer treatment strategy.

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Antitumor effect of AR3-transduced MC38 and B16F10 tumors upon irradiation in vivo. (A, D) Representative images of mice bearing mock- and AR3-transduced MC38 (A) and B16F10 (D) tumors on day 13 after tumor transplantation. Tumor regions are outlined in white (dashed elipse). Light irradiation was performed on day 6. Scale bars represent 10 mm. (B, E) Tumor growth curves of mock- and AR3-transduced MC38 (B) and B16F10 (E) tumors for 13 days after tumor transplantation. Asterisks indicate significant differences between the values (*P < 0.05; Dunnett’s test). (C, F) Body weight curves of mice bearing mock- and AR3-transduced MC38 (C) and B16F10 (F) tumors for 13 days after tumor transplantation. ″+Irr″ and ″–Irr″ indicate the presence and absence of light irradiation, respectively. The green vertical lines indicate the timing of light irradiation (60 min).

Discussion

In this study, we demonstrated that light-induced activation of AR3 effectively induces apoptotic cell death in cancer cells both in vitro and in vivo, resulting in significant tumor growth suppression without observable systemic toxicity. The ability of our method to induce apoptosis not only in HeLa cells, as previously reported, but also in MC38 and B16F10 cancer cells, suggests that the underlying mechanism is broadly applicable across multiple tumor types. Although intracellular alkalinization, the proposed trigger for apoptosis in HeLa cells, was not directly assessed in this study, it is conceivable that a similar mitochondrial-mediated apoptotic pathway is induced by intracellular alkalinization upon AR3 activation in MC38 and B16F10 cells. Our findings (ΔΨm collapse, caspase-3 activation, and DNA fragmentation, Figure ) strongly support the involvement of mitochondria-mediated apoptosis; however, confirmation by direct observation of cytochrome C release will be necessary in future studies. Some studies have suggested that intracellular alkalinization may enhance apoptotic sensitivity in cancer cells by promoting activation of BAX, which in turn leads to mitochondrial outer membrane permeabilization, cytochrome c release, and activation of the caspase cascade. − However, conformational activation of BAX was not observed during the AR3-dependent light-induced apoptosis, suggesting that a BAX-independent pathway may be involved (Figure S3). We found that ROS levels were significantly increased after irradiation in MC38 and B16F10 cells (Figure S4A), which may have contributed to mitochondrial dysfunction and the initiation of apoptosis. Interestingly, apoptosis was more readily induced in B16F10 melanoma cells than in MC38 colon cancer cells (Figure ). This heightened sensitivity may be attributable to the intrinsically elevated basal ROS levels reported in melanoma cells, which are known to rely on robust antioxidant systems to maintain redox balance. When additional ROS are generatedsuch as through optogenetically induced cytosolic alkalinization and subsequent mitochondrial dysfunction, this redox balance can be overwhelmed, leading to oxidative stress-mediated cell death. Consistently, previous studies have highlighted that melanoma cells are particularly susceptible to treatments that exacerbate ROS levels, pushing them beyond a lethal threshold. In this context, the enhanced apoptotic response observed in B16F10 cells supports the hypothesis that ROS, particularly mitochondrial ROS, is a key effector in AR3-mediated cell death. It is well established that several cancer cell types, such as glioblastoma, pancreatic ductal adenocarcinoma (PDAC) and prostate cancer, exhibit overexpression of antiapoptotic proteins and inactivation of pro-apoptotic proteins upstream of mitochondrial-mediated apoptosis pathway, leading to the apoptotic resistance and reduced efficiency of anticancer drugs. , Since the AR3-based method likely initiates mitochondrial-mediated apoptosis pathway via ROS generation, we speculate that it could be applied to such apoptosis-resistant cancers and exert potent anticancer effects. Furthermore, various cancer cell types exhibit diverse genetic mutations and alterations in signaling cascades downstream of the mitochondrial-mediated pathway, which can confer strong resistance to apoptosis. In particular, certain cancer types are highly refractory to conventional therapies and fail to undergo apoptotic cell death. Therefore, there is increasing interest in developing strategies that induce nonapoptotic forms of regulated cell death. Recent studies have shown that intracellular alkalinization can trigger a distinct form of regulated cell death termed alkaliptosis, especially in PDAC cells. In addition, alkalinization has been implicated in activating other nonapoptotic pathways, such as receptor-interacting protein kinase 3 (RIP3)-mediated necroptosis and mitochondrial inner membrane disruption leading to MPT-driven necrosis. , Investigation of these diverse cell death modalities would be important not only for broadening the applicability of our approach but also for highlighting the therapeutic potential of targeting intracellular alkalinization in cancer therapy.

In the in vivo experiments, cleaved caspase-3 signals were detected in MC38 and B16F10 tumors at depths of approximately 1.2 and 1.0 mm from the body surface, respectively (Figure ), indicating the extent to which light penetrated tumor tissue and activated AR3. Previous studies have reported that green light (532 nm) retains approximately 10% of its original intensity at a tissue depth of 1 mm from the skin surface. , Therefore, it is likely that apoptosis was induced in tumor regions receiving ≥ 10% of the irradiated light intensity. In contrast, deeper tumor regions may have received insufficient light to activate the AR3 and trigger apoptosis. To address this limitation, alternative light delivery strategies such as upconversion nanoparticles that convert deeply penetrating near-infrared light into visible light or needle-type optical fibers may enhance light accessibility to deeper tumor regions. Several studies have developed particles targeted to receptors on tumors, or even to subcellular compartments within cancer cells, which could greatly enhance the specificity of tumor cell targeting. These approaches may enable the application of this method to deep-seated tumors that are otherwise inaccessible to surface light irradiation. As an alternative, red-shifted proton pump rhodopsins could provide improved tissue penetration. Future efforts will be needed to engineer red-shifted molecules through mutagenesis and retinal analogue reconstitution as well as to identify naturally occurring red-shifted variants. Although cleaved caspase-3 signals were restricted to the surface regions of tumors, AR3-based light-induced treatment effectively suppressed tumor growth in vivo. Notably, in tumors derived from MC38 cells, a reduction in tumor volume was observed between days 10 and 13 after cell transplantation. This delayed regression may reflect not only the direct effects of apoptosis induction and inhibition of cell proliferation but also the engagement of antitumor immune responses. We observed infiltration of immune cells into apoptosis-induced AR3-transduced tumor tissues following irradiation (Figures and S10), supporting the contribution of antitumor immunity. Further analysis of activated immune cell populations using immunofluorescence or tissue cytometry will help validate this hypothesis and clarify the molecular basis for potential clinical applications in the future. MC38 cells are known to be highly immunogenic and susceptible to immune-mediated tumor rejection, resembling the tumor regression observed upon treatment with anti-PD-1 antibodies. Given the growing interest in cancer immunotherapy, it is conceivable that AR3-induced tumor cell death may act as an immunogenic stimulus capable of triggering systemic antitumor immunity. If this approach indeed facilitates immune activation, then it could serve as a promising modality in combination with immune-based therapies. Thus, our optogenetic method may represent a valuable alternative to conventional optical cancer therapies, such as photodynamic therapy (PDT) using porphyrin-based molecules , and photoimmunotherapy (PIT) employing a conjugate of a photosensitizer (IR700) and a monoclonal antibody (mAb), both of which often induce necrotic cell death. However, for clinical translation, it will be essential to establish methods for inducing AR3 expression in pre-existing tumors since in this study we injected pretransduced tumor cells, which do not fully mimic real tumor conditions. Based on our previous and current studies, we speculate that AR3-induced alkalization may also trigger cell death in nontumor cells in vivo, similar to its effect in tumor cells, although this has not yet been examined in living mice. Therefore, it is critical to ensure that the expression of AR3 is selectively and exclusively targeted to tumor cells. Currently, viral vectors such as adenoviruses and adeno-associated viruses (AAVs) are widely employed for gene delivery into tumor cells, offering distinct advantages in terms of transduction efficiency, duration of gene expression, and immunogenicity. , In addition, oncolytic viruses engineered to selectively replicate in and lyse tumor cells are under active investigation as promising platforms for both gene delivery and direct tumor destruction. , Furthermore, the use of tumor-specific promoters may further enhance the safety and specificity of AR3 expression. For future applications under real tumor conditions, it will be essential to leverage these viral platforms to develop a therapeutic strategy in which AR3 is selectively expressed in tumor tissues rather than in the surrounding normal cells, thereby enabling light-triggered induction of cancer cell apoptosis.

In summary, our study establishes an optogenetic strategy for inducing cancer cell death through AR3-mediated intracellular alkalinization. By demonstrating light-triggered apoptosis and significant tumor growth suppression in two distinct cancer models, MC38 and B16F10, we highlight the generalizability and effectiveness of this approach. The spatiotemporal precision afforded by light activation offers a unique advantage in minimizing off-target effects. To translate this platform into clinical applications, future studies should focus on achieving efficient and tumor-selective AR3 delivery, improving light penetration to reach deeper tumor tissues, and elucidating the full spectrum of cell death modalities including nonapoptotic pathways triggered by intracellular alkalinization. Furthermore, combining this method with existing cancer therapies may enhance its therapeutic potential across a broader range of tumor types.

Supplementary Material

ja5c13053_si_001.pdf (1.6MB, pdf)

Acknowledgments

We also thank Drs. Hiromu Yawo and Toru Ishizuka for providing materials including genes and vectors. This work was financially supported by JSPS KAKENHI Grant Numbers JP23K27142 to KK and JP25K22451 to YS. This research was partially supported by the Takeda Science Foundation to KK and the G-7 foundation to YS. SN was supported by JSPS Research Fellowship for Young Scientists (23KJ1613). This work was also supported by JSPS Program for Froming Japan’s Research Universities (J-PEAKS) Grant Number JPJS00420230010 to KK and YS.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c13053.

  • Additional experimental details, materials, methods, results, and references (PDF)

The authors declare no competing financial interest.

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