Photothermal-triggered non-apoptotic regulated cell death-related pathway regulation boosts systemic antitumor immunity

Abstract

Overcoming tumor cell resistance to apoptosis and immunosuppression remains a formidable challenge in tumor immunotherapy. There is a critical need to explore alternative cell death pathways to remodel the immunosuppressive tumor microenvironment (ITME), ultimately improving therapeutic efficacy. Emerging non-apoptotic regulated cell death (RCD) modalities, including ferroptosis, pyroptosis, and immunogenic cell death (ICD), have been recognized as promising strategies for tumor immunotherapy. Mild photothermal therapy (mPTT) can reshape the ITME to trigger antitumor immunity, but its clinical use is blocked by tumor cell thermotolerance. To address this, we developed a synergistic immuno-photothermal therapy within the framework of non-apoptotic RCD, aiming to modulate the ITME. It shows remarkable capabilities in catalyzing ROS generation. Under near-infrared (NIR) irradiation, ROS production was further enhanced, leading to increased lipid peroxidation (LPO) accumulation and glutathione (GSH) depletion, collectively triggering ferroptosis. Notably, heat shock proteins (HSPs) are suppressed during ferroptosis, which further amplifies the potency of mPTT to overcome the thermotolerance hurdle presented by tumor cells. Moreover, this synergistic effect activated the Caspase-1/GSDMD-dependent pathway, consequently inducing pyroptosis. The interplay between pyroptosis and ferroptosis amplified the ICD response, resulting in the release of damage-associated molecular patterns and contributing to the remodeling of the ITME. RNA sequencing also corroborated the system's involvement in pivotal gene alterations related to ferroptosis and pyroptosis. Overall, this work is expected not only to overcome tumor cell thermotolerance during mPTT but also to address long-standing challenges in tumor treatment by extensively activating non-apoptotic RCD modalities and remodeling the ITME.

Graphical abstract

Photothermal therapy modulates non-apoptotic regulated cell death pathways to enhance systemic antitumor immunity.

1. Introduction

Renal cell carcinoma (RCC), recognized as the most deadly cancer of the urinary system, continues to experience rising incidence and mortality rates across the globe [[1], [2], [3], [4]]. Current therapeutic approaches for RCC encompass surgical resection, immunotherapy, radiotherapy, molecular targeted therapy, and radiofrequency ablation. However, advanced RCC exhibits poor responsiveness to both radiotherapy and chemotherapy [5], and immunotherapy demonstrates efficacy in only a limited subset of patients [6]. Consequently, RCC is often associated with high rates of metastasis and recurrence in the absence of effective therapeutic interventions [7]. This highlights the pressing necessity to investigate and develop more effective therapeutic approaches for managing RCC. Tumor immunotherapy leverages the body's immune mechanisms to identify and eliminate cancerous cells [8]. Nevertheless, its efficacy is significantly limited by a low response rate, largely due to the tumor's limited immunogenic potential and the immunosuppressive characteristics of the tumor microenvironment (ITME) [9,10]. Tumor cells interact with various immune cells and immune components in the tumor microenvironment (TME), consequently shaping an immunosuppressive tumor microenvironment (ITME) that evades immune surveillance [[11], [12], [13]]. Additionally, tumor cells can influence the immune system to facilitate immune evasion by reducing their immunogenicity [14,15]. Furthermore, resistance to apoptotic pathways significantly hinders the treatment efficacy of tumor. Activating non-apoptotic RCD modalities and remodeling the ITME are crucial strategies to enhance the efficacy of immunotherapy in RCC.

Apoptosis is often seen as a form of cell death that does not invoke an immune response [16]. In contrast, non-apoptotic RCD types, including ferroptosis and pyroptosis, have been demonstrated to increase the immunogenicity of tumor cells when induced [17]. Ferroptosis is an iron-dependent programmed cell death mechanism driven by the excessive accumulation of reactive oxygen species (ROS), which triggers lipid peroxidation (LPO) and ultimately causes damage to the cell membrane [[18], [19], [20]]. Pyroptosis is a form of RCD mediated by the gasdermin family of proteins, marked by membrane rupture and the subsequent release of contents from within the cell [21]. Immunogenic cell death (ICD), encompassing pyroptosis, ferroptosis, and necroptosis, represents a specific form of RCD [22]. The induction of ICD encourages the secretion of damage-associated molecular patterns (DAMPs), which are essential for triggering the immune response, thereby enhancing the detection and eradication of tumor cells [23]. During ICD, DAMPs interact with immune cells and promote their recognition and phagocytosis by antigen-presenting cells (APCs). This process facilitates dendritic cell (DC) maturation and enhances T cell activation, ultimately leading to robust antitumor immune responses. This cascade ultimately activates robust antitumor immune responses to target and destroy tumor cells [24,25]. These emerging forms of regulated cell death have the potential to remodel the immunosuppressive tumor microenvironment, thereby promoting the transition of immunologically “cold” tumors into “hot” tumors with active immune responses [26].

Photothermal therapy (PTT) is an innovative tumor treatment strategy that employs photothermal agents to convert light into heat for the destruction of tumor cells. PTT can induce RCD in tumor cells, triggering ICD and consequently enhancing antitumor immunity [27,28]. However, conventional PTT typically requires high local temperatures (50–60 °C) to achieve effective tumor ablation, which may result in several adverse effects such as severe pain, skin damage, and thermal injury to surrounding healthy tissues. In contrast, mild photothermal therapy (mPTT) operates within a lower temperature range (42–45 °C), which is sufficient to induce tumor cell death while minimizing nonspecific heat diffusion and collateral damage to adjacent normal tissues [29]. In addition to its reduced side effects, previous studies have demonstrated that mPTT can stimulate systemic immune responses, thereby alleviating the ITME and offering a promising clinically relevant antitumor strategy [30]. However, tumor cells under thermal stress often initiate self-protective mechanisms by upregulating heat shock proteins (HSPs), which assist in repairing heat-induced damage and maintaining cellular homeostasis [28,31]. During photothermal therapy, this upregulation of HSPs can lead to the development of thermotolerance in tumor cells, consequently diminishing the overall therapeutic efficacy [32]. Moreover, monotherapy with PTT is often insufficient to completely eradicate tumor tissues. Combining PTT with other therapeutic modalities, such as chemotherapy, photodynamic therapy, gene therapy, or immunotherapy, has shown great potential by enabling synergistic effects that significantly enhance the overall therapeutic efficacy [33]. In recent years, nanoparticles, as a type of photothermal agent, have increasingly served as multifunctional platforms that facilitate the integrated combination of PTT with other treatment strategies [24,34]. Photothermal therapy based on AuroShell nanoparticles (sealed gold nanoshell on silica) has been approved for clinical trials for photothermal ablation of solid tumors in humans [35]. Thus, developing multifunctional nanoparticle platforms with simple synthesis methods for multimodal synergistic tumor therapy holds significant clinical translational potential and may improve the antitumor efficacy of PTT.

This study utilizes mPTT to target non-apoptotic RCD, aiming to develop a multimodal tumor treatment strategy and remodel the ITME. To this end, a multifunctional platform based on iron phthalocyanine nanoparticles (FePc NPs) has been developed to facilitate synergistic immuno-photothermal therapy (Scheme 1). The FePc NPs exhibit peroxidase-like (POD-like) activity, catalyzing the generation of ROS, particularly hydroxyl radicals (•OH), from intracellular hydrogen peroxide (H₂O₂) via the Fenton reaction. Under NIR irradiation, FePc NPs demonstrate excellent photothermal performance, which further accelerated the Fenton reaction. Under the synergistic influence of photothermal treatment, this system significantly augmented the depletion of glutathione (GSH) triggered by ROS and hastened the inactivation of glutathione peroxidase 4 (GPX4). Simultaneously, it fostered the accumulation of LPO, thereby triggering ferroptosis. Notably, HSPs are suppressed during ferroptosis, which further amplifies the potency of mPTT to overcome the thermotolerance hurdle presented by tumor cells. Additionally, this system activated the Caspase-1/GSDMD-dependent pathway to induce pyroptosis, triggering local inflammatory responses and attracting the aggregation of immune cells. The interaction between pyroptosis and ferroptosis forms a positive feedback loop, significantly amplifying the ICD response and releasing DAMPs. Subsequently, this response accelerated the maturation of dendritic cells, facilitated the polarization of macrophages, and expedited the infiltration of CD8⁺ T cells, thereby reconstructing the ITME. Finally, RNA sequencing also confirmed that this system brings about alterations in key genes associated with ferroptosis, pyroptosis and ICD. Overall, this study enhances the efficacy of mPTT by overcoming thermotolerance and achieves potent immunomodulation through extensive activation of non-apoptotic RCD pathways and remodeling of the ITME, effectively addressing key challenges in tumor therapy.

Scheme 1.

Schematic diagram of photothermal therapy modulating non-apoptotic regulated cell death-related pathway to amplify systemic antitumor immunity.

2. Results and discussions

2.1. Preparation and Characterization of FePc NPs

FePc NPs represent a novel class of photothermal nanocatalytic nanoparticles, synthesized through a conventional reverse evaporation method (Scheme 1). The nanoparticles are evenly distributed in the aqueous solution, and transmission electron microscopy (TEM) images revealed a uniform spherical shape (Fig. 1a). The analysis using dynamic light scattering (DLS) indicates that the mean diameter of FePc NPs is 87.57 nm, with a zeta potential of −44.6 mV (Fig. 1b and c). In addition, FePc NPs exhibited excellent dispersion in a range of solvents, such as H₂O, PBS, and RPMI-1640 medium, demonstrating good stability over a 7-day period across different mediums with consistent particle sizes (Fig. S1). Overall, these properties enhance the circulatory stability of FePc NPs and promote their effective infiltration into tumor tissues through the enhanced permeability and retention (EPR) effect, which is essential for in vitro and in vivo studies as well as for potential clinical applications.

Fig. 1.

Characterization of FePc NPs. (a) TEM image showcasing FePc NPs (scale bar: 100 nm). (b) The distribution of sizes for FePc NPs assessed through DLS. (c) Zeta potential measurement of FePc NPs. (d) UV–vis–NIR absorption spectrum of FePc NPs in aqueous solution. (e) Temperature variations of FePc NPs at different concentrations under NIR laser irradiation. (f) On/off heating cycles of FePc NPs (200 μg/mL) under NIR laser irradiation. (g) Photothermal images of FePc NPs at various concentrations under NIR laser irradiation. (h) Photothermal performance of FePc NPs (200 μg/mL) under NIR laser irradiation. (i) Fitted linear relationship between time and -Ln (θ). (j) Absorbance changes of TMB under different conditions. (k) Measurement of fluorescence intensity using DCFH-DA under various conditions, with excitation at 488 nm and emission at 525 nm on a fluorescence microplate reader (n = 3). (l) EPR spectra characterizing •OH signals under various conditions. The data are presented as mean ± SD. “ns” denotes no significant difference; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate levels of significance.

To assess the photothermal effect of FePc NPs, UV–Vis–NIR absorption spectra analysis was performed. The maximum absorption of FePc NPs was found to be in the range of 750–850 nm (Fig. 1d). The fluorescence intensity of FePc NPs under 800 nm and 808 nm irradiation was almost negligible (Fig. S2), indicating their efficient conversion of NIR laser irradiation into thermal energy for in vivo tumor ablation. Moreover, FePc NPs exhibited a concentration- and time-dependent increase in temperature under NIR laser irradiation (Fig. 1e and Fig. S3). To investigate the photothermal stability of FePc NPs, six heating-cooling cycles were performed, demonstrating excellent photothermal stability (Fig. 1f). As shown in Fig. 1g, the temperature of the FePc NPs solution steadily rose after continuous irradiation with an 808 nm laser (0.8 W/cm²) for 5 min, as observed by infrared thermography. Additionally, based on the temperature variation data obtained from the heating-cooling cycles and the method described in previous literature [36], the photothermal conversion efficiency (η) of FePc NPs was measured to be 56.51 % (Fig. 1h and i), further confirming that FePc NPs serve as an effective agent for photothermal tumor ablation.

Nanozymes refer to a category of nanomaterials that exhibit catalytic properties similar to those of enzymes [37]. Due to their remarkable catalytic efficacy and strong stability, nanozymes have been extensively utilized in tumor treatment, antibacterial applications, and anti-inflammatory therapies [38]. Nanozymes demonstrate POD-like activity and generate •OH through the Fenton reaction, a process that is strongly linked to their antitumor properties. We utilized 3,3′,5,5′-tetramethylbenzidine (TMB) to investigate whether FePc NPs exhibit POD-like activity. TMB is a colorless compound that, upon oxidation by ROS, transforms into a blue oxidized TMB (oxTMB) with a maximum absorption peak at 652 nm. In comparison to the other groups, the FePc NPs plus H₂O₂ group showed a significantly increased absorbance at 652 nm, indicating that FePc NPs possess POD-like catalytic activity (Fig. 1j). Based on this, we hypothesize that FePc NPs may catalyze the production of ROS from H₂O₂ in the TME, contributing to antitumor therapy. To further investigate the generation of ROS by FePc NPs, we utilized the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, measuring fluorescence intensity at 488/525 nm to quantify ROS production. The FePc NPs plus H₂O₂ group showed a marked enhancement in fluorescence intensity at 488/525 nm, confirming the production of ROS in FePc NPs solutions (Fig. 1k). Notably, NIR irradiation further increased ROS levels in the group treated with FePc NPs and H₂O₂ (Fig. S4), which may be attributed to the elevated temperature significantly accelerating the Fenton reaction. Additionally, electron paramagnetic resonance (EPR) spectroscopy was utilized to analyze the characteristic peaks of •OH in FePc NPs solutions. The FePc NPs plus H₂O₂ group showed a characteristic quartet •OH signal (aN = 14.8 G, g = 2.0058) in the EPR spectrum when 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to capture the •OH signals (Fig. 1l). FePc NPs display POD-like enzymatic activity, facilitating the production of highly cytotoxic •OH through a Fenton reaction.

2.2. In vitro antitumor activity of FePc NPs

Effective delivery of FePc NPs to tumor cells requires cellular internalization as a fundamental step. This study utilized renal cell carcinoma cells (Renca) obtained from the kidneys of male BALB/c mice diagnosed with renal cortical adenocarcinoma. To evaluate the internalization of FePc NPs by Renca cells, the cells were exposed to Cy5-labeled FePc NPs for 2, 4, and 8 h. As depicted in Fig. 2a, the red fluorescence intensity within the cells progressively increased with longer incubation times, and after 8 h of incubation, significant FePc NPs were observed around the cell nuclei. In conclusion, the results indicate that FePc NPs are internalized by Renca cells in a time-dependent manner.

Fig. 2.

In vitro cytotoxicity of photothermal therapy with FePc NPs. (a) Uptake of Cy5-labeled FePc NPs by Renca cells after incubation for various time periods (scale bar: 50 μm). (b) Intracellular ROS levels in Renca cells measured by flow cytometry under different treatments. (c) Quantification of the data in Fig. 2b (n = 3). (d) Fluorescence imaging of ROS in Renca cells was performed through DCFH-DA staining across various treatment conditions (scale bar: 100 μm). (e) Quantification of the data in Fig. 2d (n = 3). (f) Cytotoxic effects of FePc NPs at different concentrations, with or without NIR irradiation (n = 3). (g) Live/dead cell staining of Renca cells under various treatments (scale bar: 250 μm). (h) Apoptosis analysis of Renca cells under various treatments using flow cytometry. (i) Quantification of the data in Fig. 2h (n = 3). (j) The mitochondrial membrane potential of Renca cells was measured through the application of JC-1 staining under various treatment conditions. (k) Quantification of the data in Fig. 2j (n = 3). The data are presented as mean ± SD. “ns” denotes no significant difference; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate levels of significance.

The accelerated growth of tumor cells is frequently associated with increased levels of H₂O₂ in the TME, making it a promising and viable target for tumor therapy [39]. The assessment of ROS within cells was conducted through the use of flow cytometry and fluorescence microscopy, utilizing the DCFH-DA fluorescent probe. As illustrated in Fig. 2b–e, compared with the Blank group, treatment with FePc NPs alone increased intracellular ROS levels, while the combined treatment with FePc NPs and H₂O₂ further enhanced ROS generation. These findings demonstrate the catalytic activity of FePc NPs in Renca cells.

FePc NPs catalyzed the generation of ROS in Renca cells, which, at high levels, can exert antitumor effects by inducing cell death through the disruption of proteins, nucleic acids, cellular membranes, and organelles [40]. The in vitro antitumor activity of FePc NPs was assessed using the CCK-8 and the Calcein/PI Cell Viability/Cytotoxicity Assay Kit. The CCK-8 results demonstrated that FePc NPs exhibited a significant, concentration-dependent cytotoxic effect on Renca cells under NIR irradiation. In contrast, without NIR irradiation, their inhibitory effect was markedly weaker at the same concentrations (Fig. 2f). Furthermore, at higher concentrations (15 and 20 μg/mL), FePc NPs alone exhibited notable cytotoxicity, likely due to their ability to catalyze intracellular ROS generation through a Fenton reaction, ultimately leading to cell death. Upon NIR irradiation, FePc NPs induced enhanced cytotoxicity by combining photothermal effects with ROS accumulation. Similarly, Calcein-AM/PI dual staining showed consistent results with the CCK-8 assay. In comparison to both the Blank and NIR groups, the FePc NPs group displayed prominent red fluorescence (signifying dead cells) and subtle green fluorescence (indicating live cells), while the FePc NPs plus NIR group displayed the most intense red fluorescence and the least intense green fluorescence (Fig. 2g). These findings indicate that photothermal therapy with FePc NPs resulted in more effective tumor cell cytotoxicity.

Cell apoptosis was quantitatively evaluated next through Annexin V/PI staining, which was subsequently analyzed by flow cytometry. As illustrated in Fig. 2h and i, the combination of FePc NPs and NIR laser irradiation resulted in a significantly higher apoptosis rate (52.8 %) than treatment with FePc NPs alone (40.2 %). The build-up of ROS in tumor cells may result in mitochondrial dysfunction, accompanied by a decrease in mitochondrial membrane potential (MMP), which is an early indicator of apoptosis [41]. To assess changes in MMP, we used 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Red fluorescence (aggregated JC-1) indicates intact mitochondria, while green fluorescence (monomeric JC-1) reflects mitochondrial damage. Compared with other groups, the FePc NPs plus NIR group exhibited the highest proportion of green fluorescence, indicating that photothermal therapy with FePc NPs increased mitochondrial dysfunction and apoptosis (Fig. 2j and k). The observed outcomes can be linked to the enhanced POD-like activity of FePc NPs under photothermal assistance, which increases the efficiency of the Fenton reaction, resulting in increased ROS production and effectively damaging tumor cells.

In conclusion, photothermal therapy with FePc NPs results in more significant tumor cell death compared to single FePc NPs treatments. FePc NPs not only utilize nanozyme activity to catalyze ROS production, leading to tumor cell death, but also enhance tumor-killing effects through PTT, thereby demonstrating potent in vitro antitumor activity.

2.3. Mechanism of FePc NPs-Mediated cell death

Previous study has shown that the buildup of ROS can trigger multiple types of regulated cell death, including ferroptosis, pyroptosis, apoptosis, and necroptosis [42]. Apoptosis resistance is a hallmark of tumor cells, and inducing non-apoptotic RCD has become a potential therapeutic approach for tumor therapy [43]. As described earlier, photothermal therapy with FePc NPs can enhance ROS production and cell death in Renca cells, but the underlying mechanism involved is not yet fully understood. Thus, we further explored the involvement of non-apoptotic RCD pathways in the tumor cell death induced by photothermal therapy with FePc NPs.

Firstly, LPO formation was evaluated using the Liperfluo probe. When compared to the Blank group, remarkably higher levels of LPO were observed in FePc NPs plus NIR group (Fig. 3a). In addition, the quantification of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE), which are two key end products of LPO, were quantified [44]. As shown in Fig. 3b and c, MDA and 4-HNE levels increased after treatment with FePc NPs alone and were further significantly elevated combined with the NIR laser irradiation. The depletion of GSH and the inactivation of GPX4 are recognized as critical hallmarks of ferroptosis [45]. The downregulation of ferroptosis suppressor protein 1 (FSP1) and the upregulation of heme oxygenase-1 (HO-1) are also associated with ferroptosis [[46], [47], [48]]. To assess the extent of GSH depletion, we employed GSH and GSSG Assay Kit. In contrast to the Blank group, the FePc NPs group exhibited a significant reduction in GSH levels, with the most marked decrease observed in the FePc NPs plus NIR group (Fig. 3d). Additionally, we examined the expression of GPX4, FSP1, and HO-1 proteins using Western blot analysis (Fig. 3e). In comparison to the Blank group, the expression of GPX4 and FSP1 proteins was reduced in both FePc NPs with or without NIR laser irradiation, with the lowest expression observed in the FePc NPs plus NIR group, while the expression of HO-1 showed the opposite trend. The findings indicate that the addition of photothermal therapy enhances the ferroptosis induced by FePc NPs, thereby exerting an anti-tumor effect. However, after heat stimulation, HSPs are overexpressed to repair the damage inflicted on tumor cells. This self-protective mechanism confers thermotolerance to the tumor cells, thereby shielding them from the effects of PTT [31,49].

Fig. 3.

Mechanism of ferroptosis and pyroptosis induced by photothermal therapy with FePc NPs. (a) Relative levels of lipid peroxidation in Renca cells under various treatments (n = 3). (b) MDA content in Renca cells under various treatments (n = 3). (c) Protein expression of 4-HNE in Renca cells under various treatments, analyzed by western blotting. (d) Relative levels of GSH in Renca cells under various treatments (n = 3). (e) Protein expression of FSP1, HO-1, and GPX4 in Renca cells under various treatments, evaluated by western blotting. (f) The protein expression levels of HSP70 and HSP90 in Renca cells were assessed using western blotting following various treatments. (g) The protein expression of Caspase-1, cleaved Caspase-1, GSDMD, and GSDMD-N in Renca cells were assessed under various treatments using western blotting. (h) Relative levels of LDH release in Renca cells under various treatments (n = 3). (i) Relative levels of IL-1β release in Renca cells under various treatments (n = 3). (j) Schematic representation of ferroptosis/pyroptosis mechanisms induced by FePc NPs-based photothermal therapy. The data are presented as mean ± SD. “ns” denotes no significant difference; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate levels of significance.

To investigate whether photothermal therapy with FePc NPs can diminish the thermotolerance of tumor cells, Western blot was employed to evaluate the expression levels of HSP70 and HSP90 in Renca cells under different treatments. As illustrated in Fig. 3f, FePc NPs combined with NIR irradiation significantly downregulated the expression of HSP70 and HSP90 compared to the other groups. Previous studies have demonstrated that during ferroptosis, the generation of ROS and LPO end-products can inhibit HSPs by promoting their modification and degradation, thereby disrupting the self-protective mechanisms of tumor cells [31,49,50]. To verify whether this effect was directly associated with ferroptosis, Fer-1, a specific ferroptosis inhibitor, was introduced into the FePc NPs plus NIR group. The addition of Fer-1 reversed the suppression of HSP70 and HSP90 expression (Fig. S5), indicating that FePc NPs-based photothermal therapy reduces tumor thermotolerance primarily by inducing the ferroptosis pathway, ultimately enhancing the antitumor efficacy of the treatment.

Overall, these findings suggest that photothermal therapy with FePc NPs depletes intracellular GSH, inactivates GPX4, and induces LPO, thereby triggering ferroptosis in Renca cells. Notably, during the ferroptotic process, the expression of HSPs is inhibited, which disrupts the heat-protection mechanism of tumor cells, thereby enhancing the anticancer effect of ferroptosis and overcoming the thermotolerance limitations of PTT.

The generation of ROS can trigger pyroptosis, a type of RCD that is mediated by the pore-forming gasdermin (GSDM) family proteins [51,52]. The nucleotide-binding oligomerization domain (NOD)-like pyrin domain-containing receptor 3 (NLRP3) inflammasome activates Caspase-1, which subsequently cleaves gasdermin D (GSDMD) to produce its N-terminal-fragment (GSDMD-N). This segment forms openings in the cell membrane, resulting in membrane compromise and the following escape of cytoplasmic components, such as lactate dehydrogenase (LDH) and interleukin-1β (IL-1β). As shown in Fig. 3g, the combination of FePc NPs and NIR treatment significantly upregulated the expression of NLRP3, cleaved Caspase-1, and GSDMD-N proteins compared with other groups. Moreover, a significant increase in LDH (Fig. 3h) and IL-1β (Fig. 3i) concentrations was observed in the supernatant of Renca cells treated with FePc NPs combined with NIR irradiation, further confirming that photothermal therapy with FePc NPs induces pyroptosis. These findings suggest that FePc NPs combined with NIR irradiation can upregulate NLRP3, activate Caspase-1, and trigger GSDMD-dependent pyroptosis, ultimately resulting in the death of Renca cells.

As shown in Fig. S6, the presence of the ferroptosis inhibitor (Fer-1) or the pyroptosis inhibitor (Disulfiram) markedly reversed the decrease in cell viability induced by FePc NPs combined with NIR irradiation. In contrast, the addition of the apoptosis inhibitor (Z-VAD-FMK) did not significantly restore cell viability. These results further demonstrate that ferroptosis and pyroptosis are the predominant forms of cell death induced by FePc NPs-based photothermal therapy.

The potential mechanisms of photothermal therapy with FePc NPs inducing Renca cell death are shown in Fig. 3j. In combination with photothermal effects, FePc NPs further enhance the Fenton reaction to generate ROS. This photothermal therapy effectively depletes GSH, triggering LPO and subsequently inducing ferroptosis. In addition, this strategy stimulates the Caspase-1/GSDMD-dependent pathway to promote pyroptosis, triggering local inflammatory responses. Therefore, the photothermal therapy with FePc NPs exerts synergistic anticancer effects through the induction of both ferroptosis and pyroptosis.

2.4. In vitro immune response induced by FePc NPs

Tumor immunotherapy leverages the host immune system to attack tumor cells, producing antitumor effects [43]. Both ferroptosis and pyroptosis are recognized as forms of ICD [22]. Based on this, we hypothesized that photothermal therapy with FePc NPs could induce ICD, subsequently triggering the immune system and eliciting an immune response. During the ICD process, tumor cells experience distinct types of regulated cell death, such as pyroptosis and ferroptosis, leading to the release of DAMPs. The extensive release of DAMPs involves elevated calreticulin (CRT) expression, as well as the secretion of adenosine triphosphate (ATP) and high mobility group box protein 1 (HMGB1) [53]. These molecules enhance the maturation and phagocytic activity of APCs, including macrophages and dendritic cells, ultimately initiating a specific adaptive immune response [43,54]. We evaluated the intracellular levels of HMGB1 using Western blot analysis. After treatment with FePc NPs plus NIR irradiation, the intracellular HMGB1 levels were significantly reduced, suggesting that HMGB1 was relocated from the nucleus to the extracellular matrix (Fig. 4a). Additionally, extracellular ATP concentrations were determined with the use of an ATP assay kit. The ATP content increased by 3.14-fold and 3.67-fold in the FePc NPs group and FePc NPs plus NIR group, respectively, compared to the Blank group (Fig. 4b). Furthermore, the surface exposure of CRT on Renca cells was assessed. Immunofluorescence staining revealed prominent CRT fluorescence on the cell surface after treatment with FePc NPs, which was further enhanced upon laser irradiation (Fig. 4c and d). Collectively, these findings demonstrate that photothermal therapy with FePc NPs triggers ICD in tumor cells.

Fig. 4.

In vitro photothermal therapy with FePc NPs induces immunogenic cell death, macrophage polarization, and dendritic cells maturation. (a) Protein expression of HMGB1 in Renca cells under various conditions, analyzed by western blotting. (b) Relative levels of ATP release in Renca cells under various conditions (n = 3). (c) Immunofluorescence images of CRT in Renca cells under various conditions (scale bar: 50 μm). (d) Quantitative analysis related to Fig. 4c (n = 3). (e) Schematic diagram of the transwell system experiment. (f) Protein expression of iNOS and CD206 in RAW 264.7 cells under various conditions, analyzed by western blotting. (g) Immunofluorescence images of iNOS in RAW 264.7 cells under various conditions (scale bar: 20 μm). (h) Quantitative analysis related to Fig. 4g (n = 3). (i) Immunofluorescence images of CD206 in RAW 264.7 cells under various conditions (scale bar: 20 μm). (j) Quantitative analysis related to Fig. 4i (n = 3). After various treatments of RAW 264.7 cells, qPCR was employed to assess the gene expression levels of (k) CD86, (l) IL-10, and (m) Ym1 (n = 3). (n) Analysis of surface maturation markers (CD80 and CD86) on DC 2.4 cells using flow cytometry was conducted under different conditions. (o) Percentage of mature DC 2.4 cells (n = 3). The data are presented as mean ± SD. “ns” denotes no significant difference; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate levels of significance.

The presence of CRT on the cell surface is widely recognized as an “eat me” signal, which prompts APCs to phagocytose tumor cells [55]. To evaluate whether FePc NPs combined with NIR irradiation treatment can activate APCs in vitro, a transwell system was employed. In this system, Renca cells, subjected to four distinct treatments, were cultured in the upper chamber, while macrophages (RAW 264.7 cells) or dendritic cells (DC 2.4 cells) were plated in the lower chamber (Fig. 4e). Firstly, we examined the phenotypic changes in macrophages. As illustrated in Fig. 4f, compared to the Blank group, the FePc NPs plus NIR group significantly induced macrophages, with a marked upregulation of the M1 macrophage marker inducible nitric oxide synthase (iNOS) protein, while the expression of the M2 marker mannose receptor (CD206) decreased. Immunofluorescence analysis confirmed the Western blot results. Compared to the Blank group, the FePc NPs plus NIR group showed higher iNOS expression (green fluorescence) in M1 macrophages and lower CD206 expression (red fluorescence) in M2 macrophages (Fig. 4g–j). Furthermore, quantitative PCR (qPCR) analysis showed increased expression of M1-associated markers (CD86), whereas the expression of M2 markers (IL-10, Ym1) decreased (Fig. 4k–m). These results indicate that photothermal therapy with FePc NPs promotes the reprogramming of M2 macrophages into the M1 phenotype, consequently boosting the antitumor immune response. To investigate the impact of photothermal therapy with FePc NPs on dendritic cell maturation, we co-cultured Renca cells treated with four different treatments and DC 2.4 cells in the transwell system. After 24 h of co-culture, the DC 2.4 cells were harvested for flow cytometric analysis of co-stimulatory markers (CD86 and CD80) expression. The co-culture with FePc NPs plus NIR group resulted in a maturation rate of DC 2.4 cells (CD86⁺CD80⁺) as high as 17.1 %, which was notably higher than the rates observed in the other groups (Fig. 4n and o). This suggests that photothermal therapy with FePc NPs can significantly promote DC maturation.

These results collectively demonstrate that photothermal therapy with FePc NPs can induce ICD in tumor cells, while simultaneously activating APCs in vitro, thereby triggering an antitumor immune response.

2.5. Transcriptomic analysis

To investigate the mechanisms that may underlie the antitumor effects of photothermal therapy with FePc NPs, RNA was extracted from Renca cells in the Blank and FePc NPs plus NIR groups for transcriptomic analysis, aiming to elucidate differentially expressed genes (DEGs) and potential signaling pathways associated with the treatment. Principal component analysis (PCA) identified notable variations in the expression profiles between the two groups (Fig. 5a), with high correlation among the samples within each group (Fig. 5b). A total of 411 DEGs were identified based on transcriptomic changes, including 184 upregulated genes and 227 downregulated genes (Fig. 5c). Gene ontology (GO) enrichment analysis indicated that pathways related to the response to oxygen levels, metabolism, glycolysis, and ferroptosis were enriched after photothermal therapy with FePc NPs (Fig. 5d). Furthermore, gene set enrichment analysis (GSEA) showed a significant enhancement of ferroptosis and pyroptosis processes (Fig. 5e). These results further confirm the close association between FePc NPs-based photothermal therapy and both ferroptosis and pyroptosis.

Fig. 5.

RNA sequencing analysis conducted on the Blank group and FePc NPs plus NIR group. (a) Principal component analysis (PCA) results of the Blank group and FePc NPs plus NIR group. (b) The bubble plot shows the correlation analysis between the two groups. (c) The heatmap illustrates the genes with differential expression in Renca cells following various treatments. (d) The bubble plot shows the biological processes enriched in differentially expressed genes. (e) The expression of key pathways associated with FePc NPs-based photothermal therapy is indicated by gene set enrichment analysis (GSEA). (f) The heatmap illustrates the relative expression levels of genes related to RCD after FePc NPs-based photothermal therapy.

Due to the induction of ferroptosis and pyroptosis by photothermal therapy with FePc NPs, we further analyzed the relative expression levels of associated genes (Fig. 5f). Our findings indicate an overall upregulation of the gene expressions associated with both ferroptosis and pyroptosis. Given that ICD can be initiated by biological processes associated with ferroptosis and pyroptosis, we further investigated the expression levels of genes related to ICD. Similarly, the expression of ICD-related genes showed a general upregulation. The findings indicate that pathways related to non-apoptotic RCD contribute significantly to the antitumor effects of photothermal therapy with FePc NPs.

In summary, the RNA sequencing results demonstrate that photothermal therapy with FePc NPs can induce tumor cell death by activating the signaling pathways of ferroptosis, pyroptosis, and ICD.

2.6. In vivo antitumor effects of FePc NPs

To evaluate the in vivo antitumor efficacy of photothermal therapy with FePc NPs, Renca tumor-bearing mice were created by injecting Renca cells into the mice. Firstly, to investigate the tumor-targeting ability of FePc NPs in Renca tumor-bearing mice, we intravenously administered Cy5-labeled FePc NPs into the mice. The distribution of fluorescence in vivo was monitored using an in vivo imaging system at 4, 8, 12, and 24 h post-injection. As depicted in Fig. 6a, tumor tissues exhibited distinct fluorescence signals 4 h post-injection, with a progressive enhancement over time. Notably, robust fluorescence signals were detected in the tumors even 24 h post-injection. These findings suggest that FePc NPs can effectively accumulate in tumors via intravenous administration and exhibit a prolonged therapeutic window, thereby enhancing their therapeutic efficacy.

Fig. 6.

In vivo synergistic antitumor therapy. (a) In vivo fluorescence imaging at different time points after injection of Cy5-labeled FePc NPs into tumor-bearing mice. (b) Schematic timeline of in vivo treatment. (c) Thermal imaging of tumor-bearing mice in the NIR group and FePc NPs plus NIR group during NIR laser irradiation. (d) Temperature variations in the tumor area of mice in the NIR group and FePc NPs plus NIR group during NIR laser irradiation (n = 3). (e) Tumor growth curves of mice in various treatment groups throughout the course of the treatment (n = 5). (f) Body weight changes in mice in various treatment groups during treatment (n = 5). (g) Histological analysis of tumor sections post-treatment by H&E, Ki-67, TUNEL, NLRP3, CRT, and GPX4 staining (scale bar: 50 μm). (h) H&E staining, (i) Ki-67 measurement, (j) TUNEL assay, (k) NLRP3 staining, (l) CRT staining, and (m) GPX4 staining quantification of tumor tissues from various treatment groups (n = 3). (n) Relative levels of LDH in the serum of mice from different treatment groups (n = 3). (o) Relative levels of IL-1β in the serum of mice from various treatment groups (n = 3). The data are presented as mean ± SD. “ns” denotes no significant difference; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate levels of significance.

To assess the in vivo antitumor activity of FePc NPs, tumor-bearing mice were randomly allocated to one of four groups (n = 5): Blank, NIR, FePc NPs, and FePc NPs plus NIR. As illustrated in the schematic diagram (Fig. 6b), tumor-bearing mice were treated by administering 150 μL of FePc NPs (200 μg/mL) or PBS via intravenous injection on days 1 and 3. Subsequently, 24 h after each injection, the tumor regions in the NIR group and FePc NPs plus NIR group were irradiated with an 808 nm laser (0.8 W/cm²) for 10 min. To investigate the photothermal effects of FePc NPs in vivo, an infrared thermal imaging camera was utilized to record the temperature changes in the tumor regions of the NIR group and FePc NPs plus NIR group during NIR laser irradiation (Fig. 6c). As illustrated in Fig. 6d, the tumor temperature in the FePc NPs plus NIR group quickly rose to 45 °C following irradiation, whereas the temperature in the NIR group only reached 33 °C. These results indicate that FePc NPs can effectively accumulate in tumors and generate a significant photothermal effect upon NIR laser irradiation. Furthermore, this strategy employs mild photothermal therapy to eliminate tumor tissue, thereby minimizing thermal damage to adjacent healthy tissues. To evaluate the tumor suppression effects, tumor volume changes over 11 days were recorded in each group. As illustrated in Fig. 6e and Fig. S7, neither the Blank group nor the NIR group exhibited significant tumor inhibition. The FePc NPs group showed a slight inhibitory effect on tumor growth, whereas FePc NPs combined with NIR irradiation demonstrated a markedly enhanced tumor suppression effect. Throughout the treatment period, the body weights of mice across all groups stayed fairly constant, which indicates that FePc NPs exhibited negligible systemic toxicity (Fig. 6f). Furthermore, following treatment, hemolysis tests, serum biochemical analyses, and histopathological evaluations of major organs were conducted to further evaluate the biosafety of FePc NPs. The hemolysis assay demonstrated that the supernatants of FePc NPs at various concentrations appeared equally clear as those of the PBS group, with low hemolysis rates observed, confirming the absence of hemolytic activity (Fig. S8). After receiving different treatments, serum biochemical indicators in all groups of mice were within the normal range (Fig. S9), and the histopathological assessment of major organs, including the heart, liver, spleen, lungs, and kidneys, showed no significant abnormalities (Fig. S10). All evidence suggests that FePc NPs possess good biocompatibility and do not exhibit apparent systemic toxicity.

Histopathological analysis of tumor tissue sections from mice was then conducted to assess the antitumor effects of photothermal therapy with FePc NPs. We applied hematoxylin and eosin (H&E) staining to determine the extent of cellular damage and necrosis. The results showed significant necrosis and sparsely structured cells in the FePc NPs plus NIR group, partial cell necrosis in the FePc NPs group, and normal cell morphology in tumor tissues of other groups (Fig. 6g and h). Furthermore, immunohistochemical analysis of Ki-67 demonstrated the lowest proliferation level in the FePc NPs plus NIR group, with notably reduced proportion of Ki-67 positive cells when compared with the other groups (Fig. 6g and i). The findings suggest that photothermal therapy with FePc NPs significantly induces tumor cell damage and inhibits proliferation, thereby suppressing tumor growth.

We further explored the underlying mechanisms of tumor cell death in vivo. Both apoptosis and pyroptosis result in DNA fragmentation, a process that can be identified through TUNEL staining [56]. TUNEL staining results showed the strongest green fluorescence in the FePc NPs plus NIR group, indicating significant apoptosis and pyroptosis in tumors following photothermal therapy with FePc NPs (Fig. 6g and j). To validate the in vivo antitumor mechanism of FePc NPs, tumor tissue sections were analyzed using histological analysis. Immunofluorescence analysis demonstrated a notable increase in NLRP3 expression in the FePc NPs plus NIR group (Fig. 6g and k). Additionally, pyroptosis is frequently associated with the secretion of LDH and cytokines. Serum levels of LDH and IL-1β were notably higher in the FePc NPs plus NIR group when compared to the Blank group, confirming that photothermal therapy with FePc NPs induces pyroptosis (Fig. 6n and o). Furthermore, immunohistochemical analysis showed a significant downregulation of GPX4 expression in the FePc NPs plus NIR group, suggesting that photothermal therapy with FePc NPs induces ferroptosis (Fig. 6g and m). The elevated expression of CRT in the FePc NPs plus NIR group confirmed that photothermal therapy with FePc NPs induced ICD (Fig. 6g and l).

In summary, treatment with FePc NPs alone demonstrates limited antitumor efficacy. However, photothermal therapy with FePc NPs significantly enhances antitumor activity by amplifying the synergistic effects of ferroptosis, pyroptosis, and ICD.

2.7. In vivo immunomodulation of FePc NPs

Tumors are capable of evading immune surveillance via the establishment of an ITME, which consequently results in unsatisfactory immunotherapy efficacy. The objective of immunotherapy is to restrain tumor growth and reconstruct the ITME. This is achieved by activating anti-tumor immune responses and thwarting the immune escape mechanisms of tumor cells [57,58]. ICD can release DAMPs, which subsequently attract immune cells into the TME, remodeling the ITME and activating systemic immunity [[59], [60], [61]]. Therefore, inducing ICD response in tumor cells presents a promising strategy for tumor immunotherapy. Based on the in vitro and in vivo experiments, we found that photothermal therapy with FePc NPs induces ICD. Therefore, we further evaluated its potential to enhance antitumor immune responses through ICD induction. Subsequently, we performed flow cytometry analysis to assess the responses of various immune cell populations in tumor tissues following different treatments. Tumor-associated macrophages (TAMs) constitute the primary immune cell type found in the TME. Macrophages predominantly adopt the M2 phenotype in the TME, thereby facilitating tumor progression [62]. The polarization of macrophages from the pro-tumor M2 phenotype to the anti-tumor M1 phenotype is a widely adopted approach to inhibit tumor progression. Accordingly, we examined the macrophage phenotypic changes within the TME following various therapeutic interventions. Flow cytometry was employed to quantitatively assess the infiltration of M1 (F4/80⁺/CD86⁺) and M2 (F4/80⁺/CD206⁺) macrophages in the tumor. The FePc NPs plus NIR group exhibited the highest infiltration of M1 macrophages compared to other groups, with a proportion of 14.0 ± 1.4 %, which was 3.1-fold and 1.9-fold higher than that in the Blank group and the FePc NPs group, respectively (Fig. 7a and b and Fig. S12). At the same time, the percentage of M2 macrophages in the FePc NPs plus NIR group was reduced compared to the Blank group, and the M1/M2 ratio was upregulated (Fig. 7c, d and Fig. S13), which aligned with the results of macrophage polarization in vitro. These findings suggest that photothermal therapy with FePc NPs induces macrophage polarization from M2 phenotype to M1 phenotype, which consequently suppresses tumor growth.

Fig. 7.

In vivo antitumor immune responses promoted by photothermal therapy with FePc NPs. (a) Exemplary flow cytometry analysis of M1 macrophages (F4/80⁺/CD86⁺) in tumors from various treatment groups and (b) quantification (n = 3). (c) Exemplary flow cytometry analysis of M2 macrophages (F4/80⁺/CD206⁺) in tumors from various treatment groups and (d) quantification of the M1/M2 macrophage ratio (n = 3). (e) Exemplary flow cytometry analysis of mature dendritic cells (CD11c⁺CD80⁺) in tumors from various treatment groups and (f) quantification (n = 3). (g) Exemplary flow cytometry analysis of cytotoxic T cells (CD3⁺CD8⁺) in tumors from various treatment groups and (h) quantification (n = 3). (i) Exemplary flow cytometry analysis of T helper cells (CD3⁺CD4⁺) in tumors from different treatment groups and (j) quantification (n = 3). (k) Exemplary flow cytometry analysis of NK cells (CD45⁺NKp46⁺) in tumors from various treatment groups and (l) quantification (n = 3). (m) Serum IL-6 and (n) TNF-α cytokine levels in mice after various treatments (n = 3). (o) Schematic diagram of tumor microenvironment remodeling mechanism. The data are presented as mean ± SD. “ns” denotes no significant difference; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate levels of significance.

Maturation is an essential process in regulating immune responses mediated by DCs [63]. To assess the maturation status of DCs, we utilized flow cytometry to assess the expression of surface maturation markers, including the antigen-presenting molecule MHC-II and the co-stimulatory protein CD80. As shown in Fig. 7e, f and Fig. S14, the percentage of mature DCs (CD11c⁺CD80⁺) in the tumors of the FePc NPs plus NIR group was significantly higher than in the Blank group. Moreover, MHC-II expression in the FePc NPs plus NIR group was elevated to 2.7 times that of the Blank group (Figs. S11 and S15), further indicating that photothermal therapy with FePc NPs effectively promotes DCs maturation. Mature dendritic cells are proficient in antigen presentation and T cell activation, ultimately enhancing T cell infiltration into tumors [63,64]. To investigate T cell infiltration in the TME, we performed flow cytometry analysis. The proportion of cytotoxic T cells (CD3⁺CD8⁺) in the FePc NPs plus NIR group was significantly higher than that in other groups, reaching 2.7 times that of the Blank group (Fig. 7g, h and Fig. S16). Additionally, we observed a markedly elevated proportion of T helper cells (CD3⁺CD4⁺) in the FePc NPs plus NIR group compared to other groups (Fig. 7i, j and Fig. S17). The findings demonstrate that photothermal therapy with FePc NPs effectively induces substantial activation and infiltration of T cells in tumors. It is well-established that ICD also activates natural killer (NK) cells and enhances their tumoricidal activity [65]. As illustrated in Fig. 7k, l and Fig. S18, the percentage of NK cells (CD45⁺NKP46⁺) was markedly elevated in the FePc NPs plus NIR treatment group relative to the Blank group. Furthermore, FePc NPs plus NIR irradiation significantly increased the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) (Fig. 7m and n), providing additional evidence of immune response activation.

In summary, in vivo experiments demonstrated that photothermal therapy with FePc NPs effectively induced TAMs repolarization, DCs maturation, and NK cells activation, subsequently boosting T cell activation and infiltration, along with inflammatory responses characterized by increased levels of TNF-α and IL-6. All these effects collectively remodeled the ITME, activated immune responses, and ultimately inhibited tumor growth (Fig. 7o).

3. Conclusion

This study puts forth an emerging synergistic immuno-photothermal therapy within the framework of non-apoptotic RCD, aiming to modulate the ITME. In this strategy, the FePc NPs catalyzed a Fenton reaction to generate ROS that effectively killed tumor cells, and under NIR irradiation, they further enhanced ROS generation, amplified LPO accumulation, and depleted GSH, collectively triggering ferroptosis. Particularly, HSPs are suppressed during ferroptosis, thereby reducing tumor cell thermotolerance and enhancing the efficacy of mPTT. Moreover, this system activated the Caspase-1/GSDMD-dependent pathway to induce pyroptosis, triggering local inflammation. The synergistic relationship between pyroptosis and ferroptosis significantly amplified the ICD response and released DAMPs, which promoted the maturation of DCs, facilitated TAMs polarization, activated NK cells, and increased T cells infiltration. RNA sequencing confirmed it induced changes in key ferroptosis and pyroptosis-related genes, providing a molecular biology basis for the therapy. Mild photothermal therapy with FePc NPs exhibits excellent anti-tumor ability by remodeling the ITME to enhance antitumor immune responses. Collectively, this multimodal therapeutic strategy activated the non-apoptotic RCD modalities and remodeled ITME, offering an insight into overcoming suboptimal immunotherapy efficacy in RCC. Future efforts will explore the application of this therapeutic strategy to other tumor types.

4. Materials and methods

4.1. Preparation of FePc NPs

Iron (II) Phthalocyanine was initially dissolved in tetrahydrofuran (THF) at a concentration of 2 mg/mL. Subsequently, cholesterol (10 mg/mL), lecithin (20 mg/mL), DSPC (5 mg/mL), and DSPE-PEG-2000 (3.5 mg/mL) were sequentially added to the THF solution. The mixture was sonicated using a probe sonicator (50 kHz) for 20 min. Thereafter, the solution was slowly added dropwise into 10 mL of deionized water while stirring continuously for an additional 30 min. Finally, the residual THF was removed using a rotary evaporator, yielding the FePc NPs solution.

4.2. Cell culture

The Renca cell line was sourced from the American Type Culture Collection (ATCC). These cells were cultivated in RPMI-1640 medium enriched with 10 % fetal bovine serum (FBS) along with 1 % penicillin-streptomycin (Pen Strep), and they were kept at 37 °C in a humidified environment with 5 % CO₂.

4.3. Intracellular ROS detection

Renca cells were seeded into either 6-cm culture dishes or 96-well plates and subsequently kept overnight in a cell incubator. Afterward, fresh media containing 200 μM H₂O₂, 25 μg/mL FePc NPs, or a combination of 25 μg/mL FePc NPs and 200 μM H₂O₂ were added and incubated for 3 h. Following the treatment, the cells were incubated in a culture medium with DCFH-DA and kept at 37 °C for an additional 30 min. Cells cultured in 6-cm dishes were harvested and subsequently analyzed with a Beckman Coulter CytoFLEX flow cytometer, while cells in the 96-well plates were examined for green fluorescence using confocal fluorescence microscopy (Leica, TCS SP8 STED).

4.4. Cell viability assay

Renca cells were plated in 96-well plates and permitted to adhere overnight under incubation conditions. On the following day, cells were exposed to varying concentrations of FePc NPs (0, 2.5, 5, 15, and 20 μg/mL) for a duration of 8 h. A portion of the wells was then subjected to an 808 nm laser (0.8 W/cm²) for 10 min, after which they were incubated for an additional 15 h. After discarding the culture medium, 100 μL of RPMI-1640 medium containing 10 μL of CCK-8 reagent was added to each well and incubated for 2–3 h. Finally, the absorbance at 450 nm was measured using a microplate reader.

4.5. Western blotting

Renca cells were plated in 6 cm culture dishes and incubated overnight. As described previously, the cells were exposed to either RPMI-1640 medium or RPMI-1640 supplemented with FePc NPs for a duration of 8 h. After incubation, the light treatment group was exposed to NIR laser irradiation, followed by an additional 15-h incubation period. Cells from every group were gathered, subjected to lysis with RIPA buffer, and protein concentrations were assessed with a BCA assay. Identical quantities of protein from every sample were separated using SDS-PAGE and subsequently transferred to clean polyvinylidene difluoride (PVDF) membranes. The membranes underwent a 1-h treatment with 5 % non-fat milk to prevent non-specific binding, and were then incubated with primary antibodies overnight at 4 °C. After that, the membranes were exposed to the HRP-conjugated secondary antibody at room temperature for 1 h. Subsequently, the protein bands were detected using a chemiluminescence imaging system (CLiNX).

4.6. Immunofluorescence analysis

Renca cells were plated in a 96-well high-throughput plate and allowed to incubate overnight. Following this, the cells were exposed to either RPMI-1640 medium or a medium enriched with FePc NPs. After incubation, the light treatment group was exposed to NIR laser irradiation, followed by a subsequent 15-h incubation period. Following the PBS wash, the cells were treated with 4 % paraformaldehyde for fixation and then permeabilized using 0.2 % Triton X-100. The samples were subsequently treated with 5 % BSA for 30 min and then incubated overnight at 4 °C with primary antibodies targeting CRT, iNOS, and CD206. Following a wash with PBS, the cells were treated with fluorescent secondary antibodies for a duration of 1 h, and then counterstained with DAPI for 5 min. Fluorescent signals of CRT, iNOS, and CD206 were observed using confocal fluorescence microscopy (Leica, TCS SP8 STED).

4.7. Construction of the transwell system

Renca cells were grown in RPMI-1640 medium or in a medium enriched with FePc NPs. After an 8-h incubation, cells in the irradiation group underwent laser treatment, followed by an additional 15-h incubation in the cell culture chamber. At the end of the treatment, a transwell system was employed to co-culture macrophages (RAW 264.7 cells) or dendritic cells (DC 2.4 cells) with Renca cells from each group. Macrophages or dendritic cells were cultured in the lower chamber, with Renca cells positioned in the upper chamber. Subsequent evaluation of macrophage phenotype alteration and dendritic cell maturation was performed in vitro.

4.8. RNA sequencing analysis

Renca cells were plated in 6 cm culture dishes and incubated overnight. Subsequently, the cells were exposed to either standard RPMI-1640 medium or RPMI-1640 supplemented with FePc NPs for 8 h. After incubation, the FePc NPs plus NIR group was exposed to NIR laser irradiation and then incubated for an additional 15 h. Total RNA was isolated using TRIzol reagent. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer. RNA integrity was evaluated via agarose gel electrophoresis, and the RNA integrity number (RIN) was determined using an Agilent 5300 Bioanalyzer. RNA libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform. Data processing included quality control, alignment, differential gene analysis, and pathway enrichment analysis, with visualization performed using R software. Differentially expressed genes (DEGs) were identified using the R package DESeq2, with thresholds set at FoldChange >1.5 and adjusted P value < 0.05.

4.9. Construction of animal models

Experiments conducted on mice were performed in accordance with the approved protocol (AUP-211009-WJG-0002-01) by the Institutional Animal Care and Use Committee (IACUC) at Shenzhen People's Hospital, adhering to applicable national and institutional guidelines and regulations. Male BALB/c mice aged 5–6 weeks were housed in a specific pathogen-free (SPF) animal facility. Each mouse was subcutaneously injected with 100 μL of PBS containing 1.0 × 10⁶ Renca cells into the dorsal flank. Tumor size was measured with a caliper, and tumor volume was estimated using the formula: Tumor Volume=(Length × Width²)/2. After the tumor volume reached a range of 100–150 mm³, the mice were randomly assigned to different treatment groups for subsequent experiments.

4.10. In vivo antitumor treatment

The animal models were created as outlined previously, and the mice were randomly allocated into four groups (n = 5): (1) Blank group, (2) NIR group, (3) FePc NPs group, and (4) FePc NPs plus NIR group. According to the grouping, the mice received intravenous injections of 150 μL FePc NPs (200 μg/mL) or PBS on days 1 and 3. On days 2 and 4, mice in the NIR group and the FePc NPs plus NIR group were exposed to an NIR laser (0.8 W/cm², 10 min) at the tumor site. Tumor temperature variations were monitored using an infrared thermal imaging system. Tumor length, width, and body weight were regularly measured during the experiment.

4.11. Histopathological analysis

To assess the tumor tissue and major organ damage, the mice were euthanized following the treatment period, and tumor tissues, along with important organs (heart, liver, spleen, lungs, and kidneys) were harvested and preserved in 4 % PFA. H&E staining, along with immunohistochemical staining for multiple biomarkers, was carried out following the manufacturer's guidelines.

4.12. Tumor immune cell analysis

Following the completion of the in vivo treatment, tumor tissues were harvested and enzymatically digested to generate a single-cell suspension. The resulting cells were diluted and distributed into 100 μL aliquots based on the experimental needs. The cells in each group were stained with anti-CD45-PE-CF594, anti-APC-F4/80, anti-CD86-APC-CY7, and anti-CD206-PE-CY7 antibodies to assess M1/M2 macrophage polarization. For DCs analysis, cells were stained with anti-CD45-PE-CF594, anti-CD11c-PE, and anti-CD80-PE-CY7 antibodies. For the analysis of lymphocyte surface markers, antibodies targeting CD45 (PE-CF594), CD3 (FITC), CD8 (APC-CY7), and CD4 (APC) were used to stain the cells. For NK cells, anti-CD45-PE-CF594 and anti-NKp46-FITC antibodies were utilized for staining. All antibodies were incubated with the cells for 30 min at 4 °C in the dark, following the manufacturer's instructions. After washing, cells were analyzed by flow cytometry, and data were processed using FlowJo software.

4.13. Statistical analysis

The data are reported as the mean ± standard error of the mean (SEM). Statistical analyses were performed utilizing GraphPad Prism. The t-test was used to assess differences between two groups, whereas one-way analysis of variance (ANOVA) was employed to evaluate differences across multiple groups. Two-tailed t-tests were conducted to assess the statistical significance. A p-value of less than 0.05 was considered statistically significant, with “ns” indicating no significant difference. The following symbols represent the significance levels: ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

CRediT authorship contribution statement

Yanting Liang: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Haitao Yuan: Writing – review & editing, Writing – original draft, Methodology, Data curation, Conceptualization. Qiu Jin: Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing. Ali Chen: Project administration, Methodology, Formal analysis, Data curation. Wenzhe Chen: Methodology, Formal analysis, Data curation. Yunmeng Bai: Methodology, Formal analysis, Data curation. Xiaoxian Wang: Methodology, Data curation. Jingbo Ma: Methodology, Data curation. Mengyun Hou: Methodology, Data curation, Conceptualization. Yin Kwan Wong: Resources, Project administration, Data curation. Wei Xiao: Supervision, Resources, Project administration, Investigation, Formal analysis, Data curation. Xinzhou Zhang: Writing – review & editing, Resources, Project administration, Data curation. Jigang Wang: Writing – review & editing, Funding acquisition, Data curation, Conceptualization. Liping Sun: Conceptualization, Project administration, Writing – original draft, Writing – review & editing. Xijun Wang: Conceptualization, Data curation, Methodology, Project administration, Software, Writing – original draft, Writing – review & editing.

Ethics approval and consent to participate

All animal experiments conformed to the requirements of the institutional animal use and care system of Shenzhen People's Hospital (AUP-211009-WJG-0002-01).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.