Combined immune-peptide nanofiber with HSP70/AKT/mTOR axis blockade enhances near-infrared photoimmunotherapy, inhibiting tumor growth and recurrence

Combined immune-peptide nanofiber with HSP70/AKT/mTOR axis blockade enhances near-infrared photoimmunotherapy, inhibiting tumor growth and recurrence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Combined immune-peptide nanofiber with HSP70/AKT/mTOR axis blockade enhances near-infrared photoimmunotherapy, inhibiting tumor growth and recurrence Yang Du, Xinyu Zhang, Zhenqi Jiang, Wenjia Zhang, Juncheng Wu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5827209/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Although immune checkpoint blockade (ICB) therapies have shown clinical benefit, ICB remains limited effect on most “immune-cold” solid tumors, i.e., tumors with low immune infiltration. Near-infrared photoimmunotherapy (NIR-PIT) converts “cold” tumors into “hot” tumors, thereby enhancing immune responses and improving ICB efficacy. Hence, developing a strategy that can integrate NIR-PIT and ICB treatment is desirable. In this study, we designed a unique carrier-free PSP@IR-CF 3 nanofiber (NF), self-assembled from the PD-L1 targeting immune-peptide NTGYFYGDQ (PSP) and NIR-PIT agents (IR-CF 3 ). The NFs enable precise tumor targeting and immune checkpoint inhibition by specifically binding to PD-L1 on the tumor cell surface, also providing an NIR-mediated photothermal effect. Applying PSP@IR-CF 3 NFs with NIR induced mild NIR-PIT, which effectively activated the tumor immune microenvironment and treat tumors with lower immunotoxicity. Moreover, we identified that the HSP70/AKT/mTOR signaling pathway, which regulates tumor resistance and recurrence, was activated after PIT. By incorporating mTOR inhibitors like rapamycin, the combination treatment can reduce tumor resistance to NIR-PIT and decrease recurrence, thereby significantly improving therapeutic outcomes. This innovative combination therapy has the potential to revolutionize “cold” tumor treatment by offering more precise interventions that markedly enhance immunotherapeutic efficacy, reduce toxicity, and improve patient outcomes. Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles Biological sciences/Biological techniques/Nanobiotechnology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction “Cold” tumors, characterized by low immune activity and a low response rate to immunotherapy, represent a major challenge in immunotherapy 1,2 . For example, colorectal cancer exhibits a “cold” subtype, typically lacking immune cell infiltration and presenting an immunosuppressive microenvironment, making it difficult to induce a immune response and reducing the efficacy of immune checkpoint blockade (ICB) therapy 3–7 . Near-infrared photoimmunotherapy (NIR-PIT) is increasingly recognized for its precision and non-invasiveness, particularly in the treatment of immunologically “cold” tumors 1,8–11 . NIR-PIT can induce immunogenic cell death (ICD), a process that releases key tumor-associated antigens and damage-associated molecular patterns (DAMPs), converting the tumor site into an “ in situ vaccine” 12,13 . Immune cells, such as dendritic cells (DCs) and macrophages, capture and process these antigens and DAMPs, promoting effective, targeted adaptive immune responses against various solid tumors 14–16 . However, despite its promising efficacy, NIR-PIT has certain limitations. It often fails to completely inhibit tumor growth and metastasis 17,18 . This is partially due to inherent resistance mechanisms in the tumor microenvironment (TME), particularly through the upregulation of programmed cell death ligand-1 (PD-L1) under local immune activation and interferon stimulation, which triggers immune escape and diminishes the effectiveness of NIR-PIT in cancer treatment 19–21 . Therefore, combining NIR-PIT with ICB therapies, such as inhibitors targeting PD-L1 or CTLA-4, has emerged as a promising strategy 22–24 . Currently, the combination of NIR-PIT and immune checkpoint inhibitors (ICIs) typically follows a sequential protocol 22,25–28 . However, this approach may lead to complications due to intervals between treatments. After NIR-PIT, PD-L1 expression may rapidly increase in the TME, triggering immune escape and leading to tumor recurrence or metastasis, thereby impairing the efficacy of subsequent ICIs 29 . Additionally, frequent dosing may reduce patient compliance. To overcome these challenges, the co-delivery of NIR-PIT agents and ICIs using carrier-mediated drug delivery systems is being explored 30–32 . Although this strategy shows potential for synergy, challenges such as nanocarrier toxicity and uncontrolled drug release may lead to variable treatment outcomes 33 . Recent studies have shown that the self-assembly of bioactive components—particularly NIR-PIT agents and ICIs—can facilitate carrier-free delivery 34–37 . This approach enables synergistic effects with a single dose, simplifying clinical procedures, and improving treatment feasibility and patient compliance 38 . Zhao et al. developed self-delivery photo-immune stimulators (iPSs), which used Ce6 for photodynamic therapy to activate the immune microenvironment, while NLG919 inhibited IDO-1 activation, which reversed the immunosuppressive TME 39 , thereby demonstrating significant therapeutic effects and prolonging survival in tumor-bearing mouse models. These advancements underscore the potential of carrier-free photosensitizer-based nanoparticles in advanced immunotherapeutic strategies. However, such studies often rely on passive delivery, lacking the precision of active targeting strategies that leverage ligand-receptor interactions for drug-specific targeting of cancer cells, which increases off-target effects, reduces therapeutic efficacy, and elevates the risk of side effects 26,40–42 . Hence, peptides with targeting abilities, immunotherapeutic effects, and self-assembly properties have attracted increasing attention 43 . NTGYFYGDQ (PSP), a PD-L1 targeting immune peptide, possesses dual functionality: it can self-assemble into nanofibers (NFs) and simultaneously block immune checkpoints. PSP significantly enhances immune cell recognition and subsequent attacks on tumor cells by disrupting tumor immune evasion mechanisms 44 . Hence, we aim to develop a novel carrier-free PSP@IR-CF3 NF systems through self-assembling PSP with the mild NIR-PIT agent IR-CF 3 . The specific interaction between PSP and PD-L1 significantly enhances precise tumor targeting and ICB efficacy, and IR-CF 3 converts light energy into heat under NIR irradiation. Consequently, the unique design of PSP@IR-CF 3 NFs exhibits multifunctional theranostic properties and possesses significant potential for clinical applications. It is noteworthy that NIR-PIT may cause tumor therapeutic resistance and lead to tumor recurrence 45 . Hence, in this study, we performed a transcriptomic analysis, and identified that the HSP70/AKT/mTOR signaling pathway was overactivated and responsible for therapeutic resistance and potential tumor recurrence. To overcome the NIR-PIT induced resistance, we used rapamycin, a specific mTOR inhibitor, FDA-approved for use in certain cancer therapies 46–48 . Our study explored the synergistic effects of combining NIR-PIT of PSP@IR-CF 3 with rapamycin, and found that it may significantly reduce tumor recurrence rates. Overall, our study underscores the promising clinical potential of combining PSP@IR-CF 3 -NIR-PIT with rapamycin for comprehensive tumor management. Results Preparation and characterization of PSP@IR-CF 3 Using a solution-based self-assembly method, we successfully synthesized PSP@IR-CF 3 NFs. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses revealed that the NFs exhibited a uniform fibrous morphology with a thickness of approximately 2 nm, and IR-CF 3 was evenly distributed within the fibers, indicating its active participation in the co-assembly process (Fig. 2 a and Supplementary Fig. 1–3). Elemental analysis showed that fluorine (F) from IR-CF 3 was uniformly present across the fibers, suggesting that IR-CF 3 was not merely adsorbed onto PSP, but actively involved in the self-assembly process (Fig. 2 b, c). Circular dichroism (CD) spectroscopy further confirmed the fibrous nature of the assemblies, consistent with the AFM and TEM observations (Fig. 2 d). Surface plasmon resonance (SPR) experiments demonstrated that, despite the presence of IR-CF 3 , the NFs retained strong binding affinity (27.8 ± 2.2 nM) with the PD-L1 protein, highlighting their potential for targeted therapy (Supplementary Fig. 4). UV-Vis absorption spectra revealed significant absorption peaks in the near-infrared region, particularly at 808 nm and 825 nm, indicating that the NFs are suitable for photothermal therapy (Fig. 2 e). Moreover, molecular dynamics (MD) simulations were performed to further investigate the self-assembly behavior of PSP and IR-CF 3 . This dynamic process involved the movement of PSP and organic molecules, forming stable self-assembled vesicles, which maintained their structure through dynamic equilibrium (Fig. 2 f–i, Supplementary Fig. 5–6). The interactions between PSP and IR-CF 3 were primarily driven by van der Waals forces, electrostatic attractions, and π-π stacking interactions between the aromatic rings of the organic molecules, which contributed to the stability of the vesicle-like structure (Supplementary Table 1, Fig. 2 j). The above results show stable fibrous morphology, strong interactions with PD-L1, and excellent photothermal properties of the self-assembled NFs. The MD simulations further confirmed the stability and vesicle-like structure of the assemblies, with both PSP and IR-CF 3 molecules contributing to the overall stability. These NFs show significant potential for applications in photothermal and immunotherapy. NIR-PIT effects and cellular binding of PSP@IR-CF To explore the NIR-PIT characteristics of the NFs, they were laser-irradiated at 808 nm under different conditions and their temperature changes were recorded using thermography. PSP@IR-CF 3 temperature varied depending on the NF concentration, irradiation time, and laser power (Fig. 3 a–c and Supplementary Fig. 7). After 3 min irradiation (1.0 W/cm²), the maximum temperatures of PSP@IR-CF 3 and IR-CF 3 (50 µg/mL) increased by 23.11°C and 26.42°C, respectively, followed by a subsequent decline in temperature (Fig. 3 a, b). The temperature with phosphate-buffered saline (PBS) remained relatively unchanged after irradiation. Compared with the control photothermal agent IR-CF 3 , PSP@IR-CF 3 retained excellent and safe photothermal performance. As PSP@IR-CF 3 concentration increased (Fig. 3 c), the heating capability also improved. Additionally, with greater laser power, the temperature of PSP@IR-CF 3 increased correspondingly (Supplementary Fig. 7). We further examined the binding specificity of PSP@IR-CF 3 to CT26 cells in vitro . CT26 cells were incubated with PSP@IR-CF 3 , IR-CF 3 , and IR-808 for 1 h, followed by flow cytometry to assess their targeting ability (Fig. 3 d, e). The mean fluorescence intensity values of CT26 cells were 428,000 ± 9,165.15 for the PSP@IR-CF 3 /IR-808 group, 311,442 ± 21,700.83 for the aPD-L1 blocking group, 5,664 ± 1,496.25 for the IR-808 group, and 1,776 ± 259.66 for the PBS group. These results demonstrated that PSP@IR-CF 3 specifically targets and binds to PD-L1 expressed in CT26 cells. Immunofluorescence results further confirmed that PSP@IR-CF 3 /IR-808 is bound to both the cell membrane and cytoplasm of CT26 cells (Supplementary Fig. 8). These results substantiated the excellent photothermal properties and PD-L1 targeting capability of PSP@IR-CF 3 in vitro , establishing a preliminary foundation for the in vivo application of NFs. We further evaluated the therapeutic efficacy of PSP@IR-CF 3 in vitro . CT26 cells were co-incubated with IR-CF 3 and PSP@IR-CF 3 , followed by NIR irradiation (Fig. 3 f). These results indicated that irradiated groups exhibited higher cell death rates than the non-irradiated groups, showing that NIR-PIT therapy had a significant tumor cell-killing effect in vitro . NIR-PIT has recently been shown to induce ICD, which is characterized by high surface expression of calreticulin (CRT), heat shock protein 70 (HSP70), and the release of high mobility group box 1 (HMGB1) (Fig. 3 g–k). These signaling molecules are known as DAMPs and are associated with ICD effects. These effects were tested after treating CT26 cells with different NFs. The data showed that PSP@IR-CF 3 + NIR markedly increased the CRT expression of tumor cells compared to the IR-CF 3 , PSP@IR-CF 3 , and IR-CF 3 + NIR treatments (Fig. 3 g, i). The levels of HSP70 and HMGB1 secreted into the cell supernatant (Fig. 3 h, j–k) showed similar tendencies, indicating that PSP@IR-CF 3 -mediated PIT exhibits effective in vitro tumor cell-killing capability. In vivo biodistribution and NIR-PIT capacity of PSP@IR-CF 3 We also examined the in vivo biodistribution and tumor-targeting effects of PSP@IR-CF 3 /IR-808. CT26 tumor-bearing mice were injected with PSP@IR-CF 3 /IR-808 or IR-808 via the tail vein and the biodistribution of NFs was analyzed with fluorescence molecular imaging (FMI) at different time points. Both PSP@IR-CF 3 /IR-808 and IR-808 exhibited comparable fluorescence intensities (Supplementary Fig. 9). PSP@IR-CF 3 /IR-808 was systemically distributed at 0.5 h post-injection, and the signal steadily intensified in the tumor region (Fig. 4 a, b). For PSP@IR-CF 3 /IR-808, the fluorescence intensity in the tumor region reached its maximum 24 h after injection, and the fluorescence signal persisted for over 48 h post-injection. PSP@IR-CF 3 /IR-808 showed the strongest fluorescence intensity at 24 h post-injection, being approximately 1.6 and 2.5 times higher than those of the aPD-L1 blocking and IR-808 groups, respectively. In contrast, the fluorescence intensity of the aPD-L1 blocking and IR-808 groups reached its maximum at 24 h post-injection and then slightly decreased. To validate the in vivo FMI results, we dissected the tumors and major organs from mice 48 h post-injection and evaluated their fluorescence signals. In ex vivo FMI, the fluorescence signals of tumors in the PSP@IR-CF 3 /IR-808 group were stronger than those of tumors in the aPD-L1 blocking and IR-808 groups (Fig. 4 c, d). We concluded that PSP significantly increased CT26 tumor targeting and retention. These results revealed the dynamic biodistribution of NFs, which could guide subsequent therapeutic experiments. The CT26 tumor-bearing mice were treated with PBS, IR-CF 3 , or PSP@IR-CF 3 . After 24 h, the mice were exposed to 808 nm NIR irradiation (1.0 W/cm 2 ), and the temperatures were measured with a thermal imaging device (Fig. 4 e). For the PSP@IR-CF 3 + NIR group, the tumor site temperature rose by 10°C within 5 min, reaching a peak of about 46.2°C after 10 min (Fig. 4 f). However, no significant temperature increase was observed in the IR-CF 3 + NIR and PBS + NIR groups. These results confirm the in vivo NIR-PIT effects of PSP@IR-CF 3 , indicating that the temperature rise was because of improved tumor targeting and elevated accumulation of PSP@IR-CF 3 at tumor sites. In vivo NIR-PIT of PSP@IR-CF 3 We studied the anti-tumor effects of PSP@IR-CF 3 in CT26 tumor-bearing mice. The treatment scheme is shown in Fig. 5 a. Representative images of the mice at different time points following treatment are presented in Fig. 5 b. The therapeutic response was assessed by monitoring the tumor volume and weight (Fig. 5 c, d and Supplementary Fig. 10). Tumor growth was not appreciably inhibited in the PBS, IR-CF 3 , and PBS + NIR groups (Fig. 5 e–g). Mice treated with IR-CF 3 + NIR (Fig. 5 h), aPD-L1 (Fig. 5 i), or PSP@IR-CF 3 (Fig. 5 j) showed certain anti-tumor efficacy. The tumor-suppressive effect of IR-CF 3 + NIR primarily resulted from its photothermal effect. Tumor growth was reduced, but not fully eradicated, in the aPD-L1 and PSP@IR-CF3 groups, indicating that NIR or PSP@IR-CF 3 alone could not sufficiently inhibit tumor growth. However, PSP@IR-CF 3 + NIR markedly inhibited tumor growth (Fig. 5 k) and significantly extended the survival time (Fig. 5 l). Additionally, the body weights of mice in the seven groups remained stable throughout treatment (Supplementary Fig. 11). The levels of biomarkers such as ALT and AST remained within the safety thresholds (Supplementary Fig. 12). Hematoxylin and eosin staining of major organs showed no histopathological abnormalities (Supplementary Fig. 13). These findings demonstrated the biosafety and biocompatibility of this treatment. Collectively, these results suggest that PSP@IR-CF 3 can potently and safely inhibit tumor growth. NIR-PIT with PSP@IR-CF induces the ICD effect but mitigates the cytokine storm Furthermore, we examined the ability of NIR-PIT with PSP@IR-CF 3 to induce ICD in vivo . Initially, we observed that PSP@IR-CF 3 + NIR-induced programmed cell death in tumors (Supplementary Fig. 14). Subsequently, we assessed the classical markers of ICD by immunofluorescence staining of tumors from various treatment groups, revealing a significant increase in cell-surface CRT expression and extracellular release of HMGB1 in the PSP@IR-CF 3 + NIR group. In contrast, minimal expression was observed in other groups (Fig. 6 a). DCs are antigen-presenting cells crucial for initiating and regulating both innate and adaptive immunity. We therefore examined the impact of NF-induced ICD on the maturation of bone marrow-derived DCs (BMDCs) in CT26 tumor-bearing mice. After treatment, we collected and analyzed the spleens and tumors. The PSP@IR-CF 3 + NIR group had a significantly higher percentage of mature DCs compared to the other groups (Fig. 6 b, g). These data suggest that PSP@IR-CF 3 + NIR therapy effectively induces ICD and stimulates DC maturation, triggering a systemic immune response. As shown in Fig. 6 c–f and h–j, the number of CD45 + CD3 + T cells, including CD3 + CD8 + and CD3 + CD4 + T cells in splenocytes, was significantly enhanced in the PSP@IR-CF 3 + NIR group. Compared to the PBS group, the percentages of CD45 + CD3 + , CD3 + CD8 + , and CD3 + CD4 + T cells in this group showed approximately 8.9-, 27.3-, and 25.2-fold improvements, respectively (Fig. 6 h–j). Flow cytometric observations demonstrated that immune responses stimulated by NIR-PIT alone or ICB alone were limited, whereas NIR-PIT with PSP@IR-CF 3 significantly activated DC maturation and recruited T cells to exert anti-tumor effects. Furthermore, NIR-PIT of PSP@IR-CF 3 significantly elevates the levels of cytokines IL-4, TNF-α, IFN-γ, and IL-6 in vivo . Compared to the control group, the PSP@IR-CF 3 + NIR group exhibited a 2.38-fold increase in IL-4 levels (2.38 ± 0.25 vs. 1.00 ± 0.32), a 4.35-fold increase in IFN-γ levels (3.66 ± 0.06 vs. 0.84 ± 0.22), a 2.35-fold increase in IL-6 levels (2.35 ± 0.11 vs. 1.00 ± 0.19), and a 1.80-fold increase in TNF-α levels (159.68 ± 23.56 vs. 88.77 ± 0.99). Furthermore, the aPD-L1 group showed substantially elevated cytokine levels: IL-4 increased by 30.95-fold (30.95 ± 9.39 vs. 1.00 ± 0.32), IFN-γ increased by 19.82-fold (116.64 ± 8.05 vs. 0.84 ± 0.22), IL-6 increased by 11.83-fold (11.83 ± 2.12 vs. 1.00 ± 0.19), and TNF-α increased by 7.32-fold (649.39 ± 116.81 vs. 88.77 ± 0.99). Elevated levels of IL-6, IL-4, IFN-γ, and TNF-α are known to induce cytokine storms, with IL-6 serving as a key initial driver in this process (Fig. 6 k–n). Effects of combination therapy on the systemic immunological response To assess the anti-tumor efficacy of combination therapy on systemic immunological responses, mouse models with bilateral tumors were used. Once the primary tumor reached approximately 50 mm³ in volume, PSP@IR-CF 3 was intravenously administered via the tail vein. At 24 h post-injection, the primary tumors were exposed to NIR irradiation (Fig. 7 a). The results demonstrated that PSP@IR-CF 3 + NIR treatment significantly inhibited tumor growth at both the primary and distant sites, compared to the control group (Fig. 7 b–f). In the PSP@IR-CF 3 + NIR group, substantial therapeutic effects were observed on primary tumors, with significant inhibition. Distant tumors also exhibited a notable therapeutic response, indicating that PSP@IR-CF 3 + NIR had a systemic inhibitory effect. The inhibitory effect at distant tumors began to emerge 4 d post-treatment, indicating that systemic immune activation takes longer to develop than the immediate direct NIR-PIT effects observed in primary tumors. Furthermore, the group treated with PSP@IR-CF 3 alone showed moderate tumor inhibition compared with the control group, suggesting that PSP@IR-CF 3 has inherent anti-tumor properties due to its ICB capabilities. Based on this hypothesis, we analyzed immune cells within the TME of mice, focusing on T cells. The results showed significant immune activation of the TME in the PSP@IR-CF 3 + NIR group. The proportions of CD3 + CD8 + and CD3 + CD4 + T cells were markedly higher than those in the control group, with CD3 + CD8 + T cells increasing approximately 16-fold and CD3 + CD4 + T cells increasing about 3.5-fold (Fig. 7 g, i–j). In contrast, the proportion of CD4 + CD25 + Foxp3 + regulatory T cells (Tregs) decreased by approximately 6.7-fold (Fig. 7 h, k), indicating that PSP@IR-CF 3 + NIR activated the immune environment and alleviated immune suppression. This comprehensive evaluation highlighted the functionality of PSP@IR-CF 3 + NIR therapy in targeting both primary and distant tumors. The NIR-PIT with PSP@IR-CF 3 not only achieved immediate in situ tumor control but also promoted a systemic anti-tumor immune response, providing a promising approach for treating metastatic cancer. Activation of the HSP70/AKT/mTOR axis via NIR-PIT induces tumor recurrence Although NIR-PIT has shown effective therapeutic outcomes, tumor recurrence remains a significant challenge, with some tumors exhibiting resistance to NIR-PIT (Supplementary Fig. 15). To further understand the mechanism of tumor resistance, we conducted transcriptomic sequencing of recurrent tumors from the PSP@IR-CF 3 + NIR group as well as tumors from the PSP@IR-CF 3 and PBS control groups. Our analysis revealed that 20,700 genes were detected in all groups (Fig. 8 a). Volcano plots indicated that, in the PSP@IR-CF 3 + NIR group, compared to the PSP@IR-CF 3 group, 2,888 genes were upregulated, while 2,099 genes were downregulated (Fig. 8 b). Additionally, compared with the PBS group, the PSP@IR-CF 3 + NIR group had 2,657 upregulated genes and 1,729 downregulated genes, respectively (Supplementary Fig. 16). To identify the pathways potentially driving tumor recurrence, we performed a KEGG pathway analysis, which highlighted the significant upregulation of the PI3K/AKT/mTOR pathway (Fig. 8 c, d). The mTOR pathway is crucial for promoting cell proliferation which is linked to tumor recurrence. Previous research has indicated that heat shock protein 70 (HSP70) activates the mTOR pathway 49 . Given that NIR-PIT acts as a stressor that induces the expression of HSPs, including HSP70 (Fig. 8 e, f), we proposed that NIR-PIT-induced HSP70 expression upregulates the PI3K/AKT/mTOR pathway, thereby promoting tumor cell proliferation. To validate this hypothesis, we measured HSP70 expression in tumor tissues following NIR-PIT (Fig. 8 f), observing a significant increase in HSP70 protein levels. Subsequently, we knocked down HSP70 in CT26 cells and examined the PI3K/AKT/mTOR pathway, and observed a reduction in its activation (Fig. 8 g). Therefore, we conclude that the activation of the PI3K/AKT/mTOR pathway is driven by the upregulation of HSP70, leading to NIR-PIT resistance and tumor recurrence. NIR-PIT combined with mTOR inhibitor rapamycin can reduce tumor recurrence NIR-PIT can activate the immune response; however, it may also trigger cellular stress, elevate HSP70 levels, and activate the PI3K/AKT/mTOR signaling pathway, potentially resulting in therapeutic resistance in tumor cells. To address this issue, a specific mTOR inhibitor and FDA-approved drug, rapamycin, was integrated into this study to suppress the PI3K/AKT/mTOR pathway, thereby enhancing the effects of NIR-PIT and reducing recurrence rates (Fig. 9 a). In the PSP@IR-CF 3 + NIR group, 6 out of 20 mice experienced recurrence, yielding a recurrence rate of 33%. In contrast, no recurrence was observed in the 20 mice treated with PSP@IR-CF 3 + NIR combined with rapamycin (Fig. 9 b, c). To better replicate clinical conditions, patient-derived clinical samples from breast cancer patients were used to establish an in situ murine PDX model, demonstrating significant therapeutic outcomes and exceptionally high safety in the combined treatment group (Fig. 9 d, e and Supplementary Fig. 17). Figure 9 f presents ex vivo tumor images from the two PDX model groups, with significant differences in tumor weights (Fig. 9 g). These results confirmed the enhanced efficacy of this combination treatment, with significantly reduced tumor recurrence and fewer side effects. In general, NIR-PIT therapy elevates HSP70 expression, triggering the activation of the PI3K/AKT/mTOR pathway and leading to tumor recurrence. Rapamycin selectively inhibits mTOR, effectively suppressing PI3K/AKT/mTOR pathway activation, tumor recurrence, and enhancing treatment outcomes (Fig. 9 h). Discussion Clinical cancer immunotherapy faces several challenges, including low immune response rates and limited efficacy, particularly in treating “cold” tumors. To address these issues, we developed a novel carrier-free, self-assembled NIR-PIT nanodelivery system, PSP@IR-CF 3 , which integrates tumor-targeted delivery, immune checkpoint blockade and photothermal function while minimizing toxicity. The PD-L1 targeting immune-peptide PSP specifically binds to PD-L1 expressed on tumor cell surfaces, inhibiting tumor immune evasion by blocking the PD-1/PD-L1 pathway. Meanwhile, IR-CF 3 acting as a photothermal response component converts light energy into heat under NIR irradiation, activating the TME and enhancing immune responses. Unlike traditional external carrier-based delivery systems, the self-assembled, carrier-free PSP@IR-CF 3 NFs avoid issues related to nanocarrier safety, uncontrolled drug release, and inefficient targeting. This self-assembly structure improves drug biodistribution and targeting to the tumor site and extends the drug’s half-life and circulation time, reducing systemic exposure and minimizing side effects. Furthermore, PSP@IR-CF 3 enables multifaceted therapeutic effects with a single dose, increasing the combined immunotherapeutic effects with reduced toxicity, simplified treatment, and improved clinical feasibility. In terms of therapeutic efficacy, PSP@IR-CF 3 plus NIR outperforms traditional PD-L1 antibody immunotherapy. The mild photothermal therapy activates the immune microenvironment, enhances tumor tissue permeability, reduces damage to normal tissues, and promotes synergistic effects between immunotherapy and photothermal therapy 50 , 51 . PSP@IR-CF 3 plus NIR effectively induces the ICD effect, which not only directly damages tumor cells but also releases a range of immune-stimulatory molecules, such as HMGB1. These released molecules promote the activation of DCs, enhancing their antigen-presenting capacity, which induces the activation and proliferation of tumor-specific CD8 + T cells, improves the TME by increasing immune cell infiltration, and significantly boosts immunotherapeutic outcomes 50 , 52 , 53 . More importantly, unlike traditional immunotherapies, PSP@IR-CF 3 also effectively reduces the risk of cytokine storms, particularly by lowering levels of pro-inflammatory cytokines such as IL-6, IFN-γ, and TNF-α etc. Furthermore, it minimizes the severe side effects commonly associated with potent immunotherapies, thereby enhancing treatment safety 54 . Despite these promising results, NIR-PIT still encounters resistance in certain recurrent tumors, which may limit its long-term treatment efficacy. Our transcriptomic analysis identified the HSP70/AKT/mTOR pathway as a key mediator of this resistance. And we further combined PSP@IR-CF 3 with rapamycin, a selective mTOR pathway inhibitor, to overcome this challenge 55 , 56 . Rapamycin alleviates NIR-PIT-induced resistance by inhibiting the mTOR pathway, thereby reducing the risk of tumor recurrence. This combination strategy takes advantage of the synergistic effects of PSP@IR-CF 3 and rapamycin, significantly improving long-term therapeutic efficacy and prolonging treatment benefits. In summary, PSP@IR-CF 3 offers great opportunity for clinical translation due to its simple self-assembled NF structure, which significantly enhances therapeutic efficacy while reducing systemic toxicity. Its ability to deliver multi-faceted therapeutic effects with a single dose simplifies treatment regimens, improving patient compliance and efficacy. The combination with rapamycin, an FDA-approved drug, further strengthens its clinical feasibility, leveraging rapamycin’s established safety and efficacy. This approach boosts therapeutic outcomes and reduces resistance, providing a promising and effective strategy for treating “cold tumors” and advancing cancer immunotherapy. Methods Materials and reagents Peptide NQ40 (“NTKYFYEDQ”) was synthesized by Nanjing TAIYE Co. Ltd. (Nanjing, China). InVivo MAb anti-mouse PD-L1 (B7-H1) was obtained from BioX (#BE0101; Lebanon, NH, USA). The primary antibodies used include anti-calreticulin rabbit pAb (GB112134), anti-HMGB1 rabbit pAb (GB11103), and anti-HSP70 rabbit pAb (GB11241), all sourced from Servicebio (Wuhan, China). The secondary antibodies, Cy3-conjugated goat anti-rabbit IgG (GB21303) and Alexa Fluor® 488-conjugated goat anti-rabbit IgG (GB25303), were also obtained from Servicebio. Fluorochrome-conjugated anti-mouse monoclonal antibodies, including BB515 rat anti-mouse CD45 (564590; BD, Franklin Lakes, NJ, USA), APC/Fire™ 750 anti-mouse CD3 (100248; BioLegend, San Diego, CA, USA), APC anti-mouse CD4 (100412; BioLegend), PE/Cyanine7 anti-mouse CD8a (100722; BioLegend), APC/Cyanine7 anti-mouse CD11c (117324; BioLegend), PE anti-mouse CD80 (104708; BioLegend), Brilliant Violet 421™ anti-mouse CD86 (105032; BioLegend), APC anti-mouse I-A/I-E (107614; BioLegend), PE anti-mouse/human CD11b (101208; BioLegend), PE/Cyanine7 anti-mouse Ly-6G/Ly-6C (Gr-1) (108416;BioLegend), PE anti-mouse CD25 (102007; BioLegend), FOXP3 Monoclonal Antibody (FJK-16s), PerCP-Cyanine5.5 (45-5773-82; Thermo Fisher Scientific, Waltham, MA, USA), PE/Cyanine7 anti-mouse/human CD44 (103030; BioLegend), APC anti-mouse CD62L (104412; BioLegend), PE anti-mouse CD4 (116005; BioLegend), TruStain FcX™ (anti-mouse CD16/32) Antibody (101320; BioLegend). Synthesis of PSP@IR-CF 3 First, Solution A was prepared by dissolving PSP (2 mg/mL) in PBS (pH 7.4), while Solution B was made by dissolving IR-CF 3 in dimethyl sulfoxide (20 mg/mL). Solution A (2 mL) was transferred to a 300 W ultrasonic homogenizer, before 100 µL of Solution B was subjected to ultrasound treatment. The mixture was sonicated for 2 h using an ultrasonic homogenizer. The mixed solution was then removed and placed in a dialysis bag with a 1000 Da cutoff. After 12 h of dialysis in PBS solution, the liquid was removed from the dialysis membrane and stored in a 4°C refrigerator for incubation. After 24 h, the NFs were stably assembled. Cell lines CT26-Luc cells (luciferase-expressing mouse colorectal cancer cells) were sourced from the Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences (Beijing, China). CT26-Luc cells were maintained in a standard medium with 10% fetal bovine serum (FBS; Gibco, Invitrogen) and 100× penicillin–streptomycin solution (Solarbio, Beijing, China). The CT26-Luc cells were incubated at 37°C with 21% O 2 and 5% CO 2 . Animals BALB/c mice and NTG mice (NOD-scid IL2Rγ −/−), both 6 weeks old, were obtained from SPF Biotechnology Co. Ltd. (Beijing, China). Animals were kept in a specific pathogen-free (SPF) environment at 20 ± 3°C with a 12-hour light/dark cycle and free access to food and water. Animal procedures followed the guidelines for ethical research use set by Peking Union Medical College Hospital (permit number: XHDW-2022-016). Establishment of subcutaneous mouse model BALB/c mice were injected in their flanks with 1 × 10 6 CT26-Luc cells per mouse to generate the subcutaneous tumor model. In vivo photothermal immunotherapy was performed on CT26 tumor-bearing mice, which were randomly allocated to seven groups: (i) PBS, (ii) IR-CF 3 (675 µg/mL, i.v.), (iii) aPD-L1 (2 mg/mL, i.v.), (iv) PSP@IR-CF 3 (675 µg/mL, i.v.), (v) NIR (1 W/cm 2 , 10 min), (vi) CF 3 + NIR, (vii) PSP@IR-CF 3 + NIR. Bilateral CT26 tumor-bearing mice received treatment with PBS or PSP@IR-CF 3 , with or without NIR, as described previously. Establishment of PDX model Fresh human breast tumor fragments were transplanted in situ into the right mammary glands of anesthetized NTG mice. The mice were then observed in SPF conditions. Tumor growth was measured two-dimensionally using calipers. The formula for calculating the tumor volume (TV) was TV = (width 2 x length) × 0.5. The tumors were typically passaged when the TV reached 1 cm 3 . Immunogenic cell death expression assays Immunofluorescence staining was used to investigate CRT overexpression. The CT26-Luc cells were treated with PBS, CF 3 , and PSP@IR-CF 3 for 12 h, respectively. The cells were exposed to an 808 nm laser (1 W/cm 2 , 10 min) and then incubated for an additional 24 h. Subsequently, the cells were washed with PBS, fixed using 4% formaldehyde, and treated with 3% bovine serum albumin. The rabbit anti-CRT antibody at a 1:100 dilution was applied and left to incubate overnight at 4°C. After three washes with phosphate buffered saline with Tween-20 (PBST), the cells were stained with a Cy3-conjugated goat anti-rabbit secondary antibody at a 1:300 dilution, and DAPI was used to stain the nuclei. The fluorescence microscope (Nikon, Japan) was used to measure the expression level of CRT. Overexpression of HMGB1 was investigated with an enzyme-linked immunosorbent assay (ELISA) kit (SEA399Mu 96T, Cloud-clone, Wuhan, China). The CT26-Luc cells were treated with PBS, IR-CF 3, and PSP@IR-CF 3 for 12 h, respectively. The cells were exposed to an 808 nm laser (1 W/cm 2 , 10 min) and then incubated for an additional 24 h. The supernatant was spun at 4°C for 15 min at 3000 RPM and used immediately for the experiment. HMGB1 release was measured using an HMGB1 ELISA kit following the provided instructions. In vivo fluorescence molecular imaging (FMI) CT26 tumor-bearing mice were allocated into three groups. In the blocking group, 200 µg of aPD-L1 was administered intraperitoneally 12 h before imaging, and again 1 h before imaging. Mice anesthetized with isoflurane were subjected to FMI following intravenous administration of IR-808, with or without PSP@IR-CF 3 , on the IVIS Spectrum system (Perkin Elmer). Images were acquired before injection and at subsequent time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 48 h), using excitation and emission wavelengths of 745 and 800 nm, respectively. 2 d after injection, the tumor-bearing mice from each group were euthanized to retrieve the major organs (heart, liver, spleen, lung, kidney, and tumor) for imaging of the excised tissues. In vivo bioluminescence imaging (BLI) Bilateral CT26 tumor-bearing mice were monitored using BLI. Mice were anesthetized with 1.5% isoflurane in oxygen and intraperitoneally injected with D-luciferin (150 µg/g body weight; Perkin Elmer) 8 min before imaging. BLI images were acquired with the IVIS Spectrum system to monitor tumor signals at different time points after treatment in each group (pre, 4, 8, 12, and 16 d). In vivo photothermal conversion CT26 tumor-bearing mice were allocated to three groups (PBS, IR-CF 3, and PSP@IR-CF 3 ) according to the average tumor volume and injected intravenously 100 µL of IR-CF 3 and PSP@IR-CF 3 at the same concentration of 675 µg/mL. Tumors that were subjected to localized injections of PBS were used as a control group. All tumors were then irradiated with an 808 nm laser (1.0 W/cm 2 , 10min). During irradiation, the IR images of the mice were obtained with a compact thermal imaging camera (FLIR E60). After treatment, the data including tumor volumes and body weights were collected at predetermined time intervals. Evaluating anti-tumor efficacy in the CT26 tumor model The anti-tumor effects of NFs were evaluated in CT26 tumor-bearing mice. Once the tumor volumes reached around 80 mm 3 , the mice were randomly allocated into seven groups: PBS (control), IR-CF 3 , aPD-L1, PSP@IR-CF 3 , NIR, IR-CF 3 + NIR, and PSP@IR-CF 3 + NIR. Mice in the IR-CF 3 and IR-CF 3 + NIR groups received an intravenous injection of 100 µL IR-CF 3 (675 µg/mL). Mice in the PSP@IR-CF 3 and PSP@IR-CF 3 + NIR groups were injected intravenously with 100 µL PSP@IR-CF 3 (IR-CF 3 : 675 µg/mL) per mouse. NIR irradiation (1.0 W/cm 2 , 10 min) was performed 24 h after injection. Mice in the aPD-L1 groups received an intravenous injection of 100 µL aPD-L1 (2 mg/mL). Two methods were employed to determine tumor volume. One method used the formula (length × width 2 )/2, whereas the other utilized the bioluminescence intensity determined by the imaging system mentioned above. ICD expression following treatment was assessed by analyzing cleaved caspase-3, CRT, and HMGB1 levels in tumor regions using immunohistochemistry and immunofluorescence. Ex vivo analysis of T cells To perform immune cell analysis by flow cytometry, tumors or spleens from mice subjected to different treatments were harvested and stained in accordance with the provided protocols. In short, tumor cells were blocked with CD16/32 antibody (101320; BioLegend) before being stained with the following antibody combinations: CTLs and Th cells (anti-CD45-BB515, anti-CD3-APC-Fire750, anti-CD4-APC, and anti-CD8-PE-Cy7); DCs (anti-CD45-BB515, anti-CD11c-APC-Cy7, anti-CD80-PE, anti-CD86-BV421, and anti-MHCII-APC); Treg (anti-CD45-BB515, anti-CD4-APC, anti-CD25-PE, and anti-FOXP3-PerCP-Cy5.5). Finally, monoclonal antibody-stained cell suspensions were analyzed using a flow cytometer (NL-CLC3000; Cytek, Fremont, CA, USA) in accordance with standard protocols. For each test, 1 × 10 4 cells were analyzed. Cytokine detection TNF-α, IL-4, IFN-γ, and IL-6 (all from Multisciences Biotech, Hangzhou, China) in mouse serum were measured with ELISA kits following the standard protocols. Statistical analysis GraphPad Prism V10.1.2 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis. Results are shown as means ± standard error of the mean or standard deviation, as indicated. To compare two groups, Student’s t -tests were applied (P < 0.05 was deemed statistically significant). Declarations Acknowledgements This study was financially supported by Beijing Natural Science Foundation (Grants No. 7254548, 7252292, 7244524), the National Natural Science Foundation of China (Grants No. U24A20731, 81227901, 81930053, 62027901, 32101153), Young Elite Scientists Sponsorship Program by BATSA (BYESS2023244), Emerging Engineering Interdisciplinary-Young Scholars Project (PKU2024XGK007), Peking University, and Peking University Medicine Sailing Program for Young Scholars' Scientific & Technological Innovation (BMU2024YFJHPY015), the Fundamental Research Funds for the Central Universities. The authors would like to express their gratitude to Prof. Weizhi Wang from the School of Chemistry and Chemical Engineering at Beijing Institute of Technology for providing the peptide sequence, Prof. Aiguo Wu from the Ningbo Institute of Materials Technology & Engineering for synthesizing IR-CF 3 , and the Analysis & Testing Center at Beijing Institute of Technology. Author Contributions Y. D., H. X., W. H., and Z. J. conceptualized and supervised the entire research project. X. Z. authored the manuscript. Both X. Z. and Y. D. engineered and executed the imaging, therapeutic efficacy, and mechanistic studies. Z. J. and X. T. were responsible for synthesizing the materials and their characterization. W. Z. conducted in vivo imaging experiments on small animals. J. W. assisted in the collection of samples from these animals. M. S. contributed to establishing and maintaining the animal models. H. G. and C. L. participated in data analysis and further characterization of properties. Competing interests There is no conflict of interest. Data Availability All data generated or analyzed during this study are included in this published article and the Supplementary information or available from the author upon reasonable request. References Galon, J. & Bruni, D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. 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Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Supplementary information 2.Lasingreportingsummary.pdf Lasing Reporting Summary 1.Reportingsummary1.pdf Reporting Summary Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5827209","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":406676711,"identity":"99e4a6d8-c3d9-4023-aceb-656a2e04a710","order_by":0,"name":"Yang 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huadan","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2025-01-14 12:51:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5827209/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5827209/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74919797,"identity":"5c24ef04-6684-4e1e-93dc-8069c3bcae2c","added_by":"auto","created_at":"2025-01-28 10:29:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":415196,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e nanofiber (NF) development and enhanced near-infrared photoimmunotherapy (NIR-PIT) for tumor suppression and prevention of recurrence using combined PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NFs and HSP70/AKT/mTOR axis inhibition.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/faa7c3dfecf21c4e8270b7f9.png"},{"id":74919800,"identity":"4e538b18-ef3b-4496-a787-e2448f4e5c5c","added_by":"auto","created_at":"2025-01-28 10:29:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347094,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NFs. (a) Atomic force microscopy (AFM) results of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. (b) Energy dispersive spectroscopy (EDS) mapping results of C, F, N, and O. (c) EDS spectrum results of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. (d) Circular dichroism spectroscopy results of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. (e) UV absorption spectroscopy results of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. (f) Initial system setup for the assembly process of IR-CF\u003csub\u003e3\u003c/sub\u003e with PSP. (g) From the initial state to 100\u0026nbsp;ns. (h) Root mean square deviation results of PSP and IR-CF\u003csub\u003e3\u003c/sub\u003e. (i) Distance changes between PSP and IR-CF\u003csub\u003e3\u003c/sub\u003e during assembly. (j) Force analysis results of the NFs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/35d2f05f2080bb541d974fe3.png"},{"id":74920426,"identity":"56d1455d-ac8b-465d-a0a5-971b834c8763","added_by":"auto","created_at":"2025-01-28 10:37:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":291114,"visible":true,"origin":"","legend":"\u003cp\u003eNear-infrared photoimmunotherapy (NIR-PIT) effects and tumor targeting of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. (a) Temperature variations at different time points during NIR irradiation of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, IR-CF\u003csub\u003e3\u003c/sub\u003e, and PBS control groups. The statistical analysis is presented in (b), where ΔTemperature = temperature at different time points - initial temperature. (c) Temperature changes at different time points for varying concentrations of PSP@IR-CF\u003csub\u003e3 \u003c/sub\u003eunder NIR irradiation. (d, e) Flow cytometry analysis showing the targeting specificity of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 towards CT26 cells (n=3). (f) \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity of CT26 cells when incubated with increasing concentrations of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e with or without NIR irradiation. (g, h) Immunofluorescence staining showing the expression of CRT and HSP70. (i, j) Mean Fluorescence Intensity (MFI) of CRT and HSP70 immunofluorescence staining (n=7). (k) Enzyme-linked immunosorbent assay results showing the concentration of HMGB1 released by tumor cells into the culture medium (n=3). Scale bars: 50\u0026nbsp;μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/6de5e18dee129f5c86a9cac0.png"},{"id":74920428,"identity":"fd6cb12e-087f-4152-8c82-07bd4da075f2","added_by":"auto","created_at":"2025-01-28 10:37:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":413912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e tumor targeting and NIR-PIT effects of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. (a, b) Fluorescence intensity of PSP@IR-CF\u003csub\u003e3 \u003c/sub\u003ebiodistribution in CT26 tumor-bearing mice (n=3). (c, d) \u003cem\u003eEx vivo\u003c/em\u003e fluorescence imaging of organs from each group of mice at 48\u0026nbsp;h post-treatment. Fluorescence intensity of each organ was quantified and statistically analyzed (n=3). (e) Evaluation of the \u003cem\u003ein vivo\u003c/em\u003e photothermal performance of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. Statistical results are detailed in (f), with ΔTemperature = temperature at different time points - initial temperature (n=3).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/f8c5e4a9c697f160a548f61a.png"},{"id":74919813,"identity":"8c5a5546-c6f0-4857-9078-7ce28cf6ce7c","added_by":"auto","created_at":"2025-01-28 10:29:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":387213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e NIR-PIT of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. (a) Schematic illustration of the \u003cem\u003ein vivo\u003c/em\u003e PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NIR-PIT in CT26 tumor-bearing mice. (b) Representative images of mice at different time points with different treatments. (c) \u003cem\u003eEx vivo\u003c/em\u003e images of excised tumors from the treated mice. (d) Tumor volumes of different treatment groups (n=5). (e–k) Tumor volume changes in individual mice during the treatments (n=5). (l) Survival analysis of different treatment groups (n=5).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/1f3c28c1ef984c64df3bd819.png"},{"id":74919809,"identity":"9d65bf57-54cc-45d2-8c70-c6913c26764d","added_by":"auto","created_at":"2025-01-28 10:29:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":347775,"visible":true,"origin":"","legend":"\u003cp\u003eNIR-PIT of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e induces ICD, thereby activating the immune response \u003cem\u003ein vivo\u003c/em\u003e. (a) Immunohistochemical and immunofluorescence staining were used to detect the apoptotic marker cleaved caspase-3 and ICD markers CRT and HMGB1. (b) CD80\u003csup\u003e+\u003c/sup\u003e and CD86\u003csup\u003e+\u003c/sup\u003e expressions on the surface of dendritic cells (DCs). (c) CD3\u003csup\u003e+\u003c/sup\u003e expressions on the surface of T cells. (d) Populations of CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells. (e) Populations of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells. (f) Populations of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells. (g) Proportion of mature DCs (n=3). (h) Proportion of CD3\u003csup\u003e+\u003c/sup\u003e T cells (n=3). (i) Proportion of cytotoxic T lymphocyte (CTL) cells among CD3\u003csup\u003e+\u003c/sup\u003e T cells (n=3). (j) The proportion of helper T (Th) cells among CD3\u003csup\u003e+\u003c/sup\u003e T cells (n=3). (k–n) Serum concentrations of cytokines IL-4 (k), IL-6 (l), IFN-γ (m), and TNF-α (n) (n=3). Scale bars: 20 µm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/7e42b536801d1e16a1c7e1dc.png"},{"id":74919819,"identity":"d06fa722-a18b-4a6a-92f3-75c6a863afa9","added_by":"auto","created_at":"2025-01-28 10:29:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":288979,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of NIR-PIT on distant tumors and the immunological response. (a) Schematic illustration of the \u003cem\u003ein vivo\u003c/em\u003e PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NIR-PIT protocol in the bilateral tumor mice. (b) Representative images of the bilateral tumor mice at different time points after different treatments. (c–f) Statistical analysis of tumor volume and bioluminescence imaging signal intensities in different treatment groups (n=5). (g–k) Immunomodulatory effects of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e + NIR. Statistical analysis of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e CTLs (g, i), CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e Th cells (g, j), and CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Tregs (h, k) in different treatment groups (n=3).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/e027435f7b70ba63d0d2bdf9.png"},{"id":74919821,"identity":"78a5cc38-bc73-44de-9af7-48dbf8974384","added_by":"auto","created_at":"2025-01-28 10:29:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":284095,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism analysis of tumor recurrence after NIR-PIT of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e by RNA sequencing. (a) Venn map of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e + NIR, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, and PBS group. (b) The volcano plot of differentially expressed genes between PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e + NIR and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e group. (c, d) Kyoto Encyclopedia of Genes and Genomes enrichment in PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e + NIR and PBS groups (red arrows represent HSP70/AKT/mTOR relative pathways). (e) Heatmap of HSPs and PI3K/AKT/mTOR expression levels in PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e + NIR, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e,\u003csub\u003e \u003c/sub\u003eand PBS groups. (f) Western blotting of HSP70. (g) Western blotting of HSP70 and PI3K/AKT/mTOR.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/5bd0c4817baf39a31f59062c.png"},{"id":74919823,"identity":"fd658ba9-e8f4-4023-8f36-0c2f6e123e84","added_by":"auto","created_at":"2025-01-28 10:29:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":374729,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic efficacy of the combination treatment with PSP@IR-CF\u003csub\u003e3 \u003c/sub\u003e+ NIR and rapamycin. (a) Schematic illustration of PSP@IR-CF\u003csub\u003e3 \u003c/sub\u003e+ NIR\u003csub\u003e \u003c/sub\u003eand rapamycin treatment protocol in the subcutaneous CT26 and patient-derived xenograft (PDX) mouse model. (b) Representative images of the subcutaneous mouse tumor models at different time points after different treatments. (c) Tumor volume measurements in the subcutaneous CT26 mouse models on Day 28 (n=20). (d) Representative images of PDX mouse models at different time points after different treatments. (e) Tumor volume measurements in PDX mouse models on Day 28 (n=8). (f) \u003cem\u003eEx vivo\u003c/em\u003e images of excised tumors from the treated PDX mouse models. (g) Tumor weight measurements in PDX mouse models. (h) Mechanism of the combined therapy in inhibiting tumor growth and recurrence. Rapa, rapamycin.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/574bef404924ad8e3e567a35.png"},{"id":89572126,"identity":"89facfa7-d2c7-45aa-8d97-570320506ac6","added_by":"auto","created_at":"2025-08-21 12:26:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4075808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/ec8e6308-00e7-46ae-8f93-099cb36c743c.pdf"},{"id":74920427,"identity":"e52a951d-a667-4b52-9662-129668e75b56","added_by":"auto","created_at":"2025-01-28 10:37:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4587435,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/ceea1607ae42c6d9aca7251e.docx"},{"id":74919798,"identity":"927c66c8-fbf3-4dec-9c09-c7885eca8f7c","added_by":"auto","created_at":"2025-01-28 10:29:38","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":928618,"visible":true,"origin":"","legend":"Lasing Reporting Summary","description":"","filename":"2.Lasingreportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/621756c8ff92bce579248875.pdf"},{"id":74919807,"identity":"df5fdc4a-a5ae-4a7c-8fda-977bc84cb019","added_by":"auto","created_at":"2025-01-28 10:29:39","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4350273,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"1.Reportingsummary1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5827209/v1/2fd4798d79b1e15c8a291bca.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Combined immune-peptide nanofiber with HSP70/AKT/mTOR axis blockade enhances near-infrared photoimmunotherapy, inhibiting tumor growth and recurrence","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u0026ldquo;Cold\u0026rdquo; tumors, characterized by low immune activity and a low response rate to immunotherapy, represent a major challenge in immunotherapy\u003csup\u003e1,2\u003c/sup\u003e. For example, colorectal cancer exhibits a \u0026ldquo;cold\u0026rdquo; subtype, typically lacking immune cell infiltration and presenting an immunosuppressive microenvironment, making it difficult to induce a immune response and reducing the efficacy of immune checkpoint blockade (ICB) therapy\u003csup\u003e3\u0026ndash;7\u003c/sup\u003e. Near-infrared photoimmunotherapy (NIR-PIT) is increasingly recognized for its precision and non-invasiveness, particularly in the treatment of immunologically \u0026ldquo;cold\u0026rdquo; tumors\u003csup\u003e1,8\u0026ndash;11\u003c/sup\u003e. NIR-PIT can induce immunogenic cell death (ICD), a process that releases key tumor-associated antigens and damage-associated molecular patterns (DAMPs), converting the tumor site into an \u0026ldquo;\u003cem\u003ein situ\u003c/em\u003e vaccine\u0026rdquo;\u003csup\u003e12,13\u003c/sup\u003e. Immune cells, such as dendritic cells (DCs) and macrophages, capture and process these antigens and DAMPs, promoting effective, targeted adaptive immune responses against various solid tumors\u003csup\u003e14\u0026ndash;16\u003c/sup\u003e. However, despite its promising efficacy, NIR-PIT has certain limitations. It often fails to completely inhibit tumor growth and metastasis\u003csup\u003e17,18\u003c/sup\u003e. This is partially due to inherent resistance mechanisms in the tumor microenvironment (TME), particularly through the upregulation of programmed cell death ligand-1 (PD-L1) under local immune activation and interferon stimulation, which triggers immune escape and diminishes the effectiveness of NIR-PIT in cancer treatment\u003csup\u003e19\u0026ndash;21\u003c/sup\u003e. Therefore, combining NIR-PIT with ICB therapies, such as inhibitors targeting PD-L1 or CTLA-4, has emerged as a promising strategy\u003csup\u003e22\u0026ndash;24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, the combination of NIR-PIT and immune checkpoint inhibitors (ICIs) typically follows a sequential protocol\u003csup\u003e22,25\u0026ndash;28\u003c/sup\u003e. However, this approach may lead to complications due to intervals between treatments. After NIR-PIT, PD-L1 expression may rapidly increase in the TME, triggering immune escape and leading to tumor recurrence or metastasis, thereby impairing the efficacy of subsequent ICIs\u003csup\u003e29\u003c/sup\u003e. Additionally, frequent dosing may reduce patient compliance. To overcome these challenges, the co-delivery of NIR-PIT agents and ICIs using carrier-mediated drug delivery systems is being explored\u003csup\u003e30\u0026ndash;32\u003c/sup\u003e. Although this strategy shows potential for synergy, challenges such as nanocarrier toxicity and uncontrolled drug release may lead to variable treatment outcomes\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent studies have shown that the self-assembly of bioactive components\u0026mdash;particularly NIR-PIT agents and ICIs\u0026mdash;can facilitate carrier-free delivery\u003csup\u003e34\u0026ndash;37\u003c/sup\u003e. This approach enables synergistic effects with a single dose, simplifying clinical procedures, and improving treatment feasibility and patient compliance\u003csup\u003e38\u003c/sup\u003e. Zhao et al. developed self-delivery photo-immune stimulators (iPSs), which used Ce6 for photodynamic therapy to activate the immune microenvironment, while NLG919 inhibited IDO-1 activation, which reversed the immunosuppressive TME\u003csup\u003e39\u003c/sup\u003e, thereby demonstrating significant therapeutic effects and prolonging survival in tumor-bearing mouse models. These advancements underscore the potential of carrier-free photosensitizer-based nanoparticles in advanced immunotherapeutic strategies. However, such studies often rely on passive delivery, lacking the precision of active targeting strategies that leverage ligand-receptor interactions for drug-specific targeting of cancer cells, which increases off-target effects, reduces therapeutic efficacy, and elevates the risk of side effects\u003csup\u003e26,40\u0026ndash;42\u003c/sup\u003e. Hence, peptides with targeting abilities, immunotherapeutic effects, and self-assembly properties have attracted increasing attention\u003csup\u003e43\u003c/sup\u003e. NTGYFYGDQ (PSP), a PD-L1 targeting immune peptide, possesses dual functionality: it can self-assemble into nanofibers (NFs) and simultaneously block immune checkpoints. PSP significantly enhances immune cell recognition and subsequent attacks on tumor cells by disrupting tumor immune evasion mechanisms\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHence, we aim to develop a novel carrier-free PSP@IR-CF3 NF systems through self-assembling PSP with the mild NIR-PIT agent IR-CF\u003csub\u003e3\u003c/sub\u003e. The specific interaction between PSP and PD-L1 significantly enhances precise tumor targeting and ICB efficacy, and IR-CF\u003csub\u003e3\u003c/sub\u003e converts light energy into heat under NIR irradiation. Consequently, the unique design of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NFs exhibits multifunctional theranostic properties and possesses significant potential for clinical applications. It is noteworthy that NIR-PIT may cause tumor therapeutic resistance and lead to tumor recurrence\u003csup\u003e45\u003c/sup\u003e. Hence, in this study, we performed a transcriptomic analysis, and identified that the HSP70/AKT/mTOR signaling pathway was overactivated and responsible for therapeutic resistance and potential tumor recurrence. To overcome the NIR-PIT induced resistance, we used rapamycin, a specific mTOR inhibitor, FDA-approved for use in certain cancer therapies\u003csup\u003e46\u0026ndash;48\u003c/sup\u003e. Our study explored the synergistic effects of combining NIR-PIT of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e with rapamycin, and found that it may significantly reduce tumor recurrence rates. Overall, our study underscores the promising clinical potential of combining PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e-NIR-PIT with rapamycin for comprehensive tumor management.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and characterization of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eUsing a solution-based self-assembly method, we successfully synthesized PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NFs. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses revealed that the NFs exhibited a uniform fibrous morphology with a thickness of approximately 2 nm, and IR-CF\u003csub\u003e3\u003c/sub\u003e was evenly distributed within the fibers, indicating its active participation in the co-assembly process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;1\u0026ndash;3). Elemental analysis showed that fluorine (F) from IR-CF\u003csub\u003e3\u003c/sub\u003e was uniformly present across the fibers, suggesting that IR-CF\u003csub\u003e3\u003c/sub\u003e was not merely adsorbed onto PSP, but actively involved in the self-assembly process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). Circular dichroism (CD) spectroscopy further confirmed the fibrous nature of the assemblies, consistent with the AFM and TEM observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Surface plasmon resonance (SPR) experiments demonstrated that, despite the presence of IR-CF\u003csub\u003e3\u003c/sub\u003e, the NFs retained strong binding affinity (27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 nM) with the PD-L1 protein, highlighting their potential for targeted therapy (Supplementary Fig.\u0026nbsp;4). UV-Vis absorption spectra revealed significant absorption peaks in the near-infrared region, particularly at 808 nm and 825 nm, indicating that the NFs are suitable for photothermal therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Moreover, molecular dynamics (MD) simulations were performed to further investigate the self-assembly behavior of PSP and IR-CF\u003csub\u003e3\u003c/sub\u003e. This dynamic process involved the movement of PSP and organic molecules, forming stable self-assembled vesicles, which maintained their structure through dynamic equilibrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u0026ndash;i, Supplementary Fig.\u0026nbsp;5\u0026ndash;6). The interactions between PSP and IR-CF\u003csub\u003e3\u003c/sub\u003e were primarily driven by van der Waals forces, electrostatic attractions, and π-π stacking interactions between the aromatic rings of the organic molecules, which contributed to the stability of the vesicle-like structure (Supplementary Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). The above results show stable fibrous morphology, strong interactions with PD-L1, and excellent photothermal properties of the self-assembled NFs. The MD simulations further confirmed the stability and vesicle-like structure of the assemblies, with both PSP and IR-CF\u003csub\u003e3\u003c/sub\u003e molecules contributing to the overall stability. These NFs show significant potential for applications in photothermal and immunotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNIR-PIT effects and cellular binding of PSP@IR-CF\u003c/h3\u003e\n\u003cp\u003eTo explore the NIR-PIT characteristics of the NFs, they were laser-irradiated at 808 nm under different conditions and their temperature changes were recorded using thermography. PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e temperature varied depending on the NF concentration, irradiation time, and laser power (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;c and Supplementary Fig.\u0026nbsp;7). After 3 min irradiation (1.0 W/cm\u0026sup2;), the maximum temperatures of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e and IR-CF\u003csub\u003e3\u003c/sub\u003e (50 \u0026micro;g/mL) increased by 23.11\u0026deg;C and 26.42\u0026deg;C, respectively, followed by a subsequent decline in temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). The temperature with phosphate-buffered saline (PBS) remained relatively unchanged after irradiation. Compared with the control photothermal agent IR-CF\u003csub\u003e3\u003c/sub\u003e, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e retained excellent and safe photothermal performance. As PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e concentration increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), the heating capability also improved. Additionally, with greater laser power, the temperature of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e increased correspondingly (Supplementary Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eWe further examined the binding specificity of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e to CT26 cells \u003cem\u003ein vitro\u003c/em\u003e. CT26 cells were incubated with PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, IR-CF\u003csub\u003e3\u003c/sub\u003e, and IR-808 for 1 h, followed by flow cytometry to assess their targeting ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). The mean fluorescence intensity values of CT26 cells were 428,000\u0026thinsp;\u0026plusmn;\u0026thinsp;9,165.15 for the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 group, 311,442\u0026thinsp;\u0026plusmn;\u0026thinsp;21,700.83 for the aPD-L1 blocking group, 5,664\u0026thinsp;\u0026plusmn;\u0026thinsp;1,496.25 for the IR-808 group, and 1,776\u0026thinsp;\u0026plusmn;\u0026thinsp;259.66 for the PBS group. These results demonstrated that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e specifically targets and binds to PD-L1 expressed in CT26 cells. Immunofluorescence results further confirmed that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 is bound to both the cell membrane and cytoplasm of CT26 cells (Supplementary Fig.\u0026nbsp;8). These results substantiated the excellent photothermal properties and PD-L1 targeting capability of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e \u003cem\u003ein vitro\u003c/em\u003e, establishing a preliminary foundation for the \u003cem\u003ein vivo\u003c/em\u003e application of NFs.\u003c/p\u003e \u003cp\u003eWe further evaluated the therapeutic efficacy of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e \u003cem\u003ein vitro\u003c/em\u003e. CT26 cells were co-incubated with IR-CF\u003csub\u003e3\u003c/sub\u003e and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, followed by NIR irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). These results indicated that irradiated groups exhibited higher cell death rates than the non-irradiated groups, showing that NIR-PIT therapy had a significant tumor cell-killing effect \u003cem\u003ein vitro\u003c/em\u003e. NIR-PIT has recently been shown to induce ICD, which is characterized by high surface expression of calreticulin (CRT), heat shock protein 70 (HSP70), and the release of high mobility group box 1 (HMGB1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u0026ndash;k). These signaling molecules are known as DAMPs and are associated with ICD effects. These effects were tested after treating CT26 cells with different NFs. The data showed that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR markedly increased the CRT expression of tumor cells compared to the IR-CF\u003csub\u003e3\u003c/sub\u003e, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, and IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, i). The levels of HSP70 and HMGB1 secreted into the cell supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, j\u0026ndash;k) showed similar tendencies, indicating that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e-mediated PIT exhibits effective \u003cem\u003ein vitro\u003c/em\u003e tumor cell-killing capability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ebiodistribution and NIR-PIT capacity of PSP@IR-CF\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eWe also examined the \u003cem\u003ein vivo\u003c/em\u003e biodistribution and tumor-targeting effects of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808. CT26 tumor-bearing mice were injected with PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 or IR-808 via the tail vein and the biodistribution of NFs was analyzed with fluorescence molecular imaging (FMI) at different time points. Both PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 and IR-808 exhibited comparable fluorescence intensities (Supplementary Fig.\u0026nbsp;9). PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 was systemically distributed at 0.5 h post-injection, and the signal steadily intensified in the tumor region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). For PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808, the fluorescence intensity in the tumor region reached its maximum 24 h after injection, and the fluorescence signal persisted for over 48 h post-injection. PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 showed the strongest fluorescence intensity at 24 h post-injection, being approximately 1.6 and 2.5 times higher than those of the aPD-L1 blocking and IR-808 groups, respectively. In contrast, the fluorescence intensity of the aPD-L1 blocking and IR-808 groups reached its maximum at 24 h post-injection and then slightly decreased. To validate the \u003cem\u003ein vivo\u003c/em\u003e FMI results, we dissected the tumors and major organs from mice 48 h post-injection and evaluated their fluorescence signals. In \u003cem\u003eex vivo\u003c/em\u003e FMI, the fluorescence signals of tumors in the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e/IR-808 group were stronger than those of tumors in the aPD-L1 blocking and IR-808 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). We concluded that PSP significantly increased CT26 tumor targeting and retention. These results revealed the dynamic biodistribution of NFs, which could guide subsequent therapeutic experiments.\u003c/p\u003e \u003cp\u003eThe CT26 tumor-bearing mice were treated with PBS, IR-CF\u003csub\u003e3\u003c/sub\u003e, or PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e. After 24 h, the mice were exposed to 808 nm NIR irradiation (1.0 W/cm\u003csup\u003e2\u003c/sup\u003e), and the temperatures were measured with a thermal imaging device (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). For the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group, the tumor site temperature rose by 10\u0026deg;C within 5 min, reaching a peak of about 46.2\u0026deg;C after 10 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). However, no significant temperature increase was observed in the IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR and PBS\u0026thinsp;+\u0026thinsp;NIR groups. These results confirm the \u003cem\u003ein vivo\u003c/em\u003e NIR-PIT effects of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, indicating that the temperature rise was because of improved tumor targeting and elevated accumulation of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e at tumor sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eNIR-PIT of PSP@IR-CF\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eWe studied the anti-tumor effects of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e in CT26 tumor-bearing mice. The treatment scheme is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. Representative images of the mice at different time points following treatment are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb. The therapeutic response was assessed by monitoring the tumor volume and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d and Supplementary Fig.\u0026nbsp;10). Tumor growth was not appreciably inhibited in the PBS, IR-CF\u003csub\u003e3\u003c/sub\u003e, and PBS\u0026thinsp;+\u0026thinsp;NIR groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u0026ndash;g). Mice treated with IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), aPD-L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei), or PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej) showed certain anti-tumor efficacy. The tumor-suppressive effect of IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR primarily resulted from its photothermal effect. Tumor growth was reduced, but not fully eradicated, in the aPD-L1 and PSP@IR-CF3 groups, indicating that NIR or PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e alone could not sufficiently inhibit tumor growth. However, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR markedly inhibited tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek) and significantly extended the survival time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el). Additionally, the body weights of mice in the seven groups remained stable throughout treatment (Supplementary Fig.\u0026nbsp;11). The levels of biomarkers such as ALT and AST remained within the safety thresholds (Supplementary Fig.\u0026nbsp;12). Hematoxylin and eosin staining of major organs showed no histopathological abnormalities (Supplementary Fig.\u0026nbsp;13). These findings demonstrated the biosafety and biocompatibility of this treatment. Collectively, these results suggest that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e can potently and safely inhibit tumor growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eNIR-PIT with PSP@IR-CF induces the ICD effect but mitigates the cytokine storm\u003c/h3\u003e\n\u003cp\u003eFurthermore, we examined the ability of NIR-PIT with PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e to induce ICD \u003cem\u003ein vivo\u003c/em\u003e. Initially, we observed that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR-induced programmed cell death in tumors (Supplementary Fig.\u0026nbsp;14). Subsequently, we assessed the classical markers of ICD by immunofluorescence staining of tumors from various treatment groups, revealing a significant increase in cell-surface CRT expression and extracellular release of HMGB1 in the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group. In contrast, minimal expression was observed in other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eDCs are antigen-presenting cells crucial for initiating and regulating both innate and adaptive immunity. We therefore examined the impact of NF-induced ICD on the maturation of bone marrow-derived DCs (BMDCs) in CT26 tumor-bearing mice. After treatment, we collected and analyzed the spleens and tumors. The PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group had a significantly higher percentage of mature DCs compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, g). These data suggest that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR therapy effectively induces ICD and stimulates DC maturation, triggering a systemic immune response. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u0026ndash;f and h\u0026ndash;j, the number of CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells, including CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells in splenocytes, was significantly enhanced in the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group. Compared to the PBS group, the percentages of CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e, and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells in this group showed approximately 8.9-, 27.3-, and 25.2-fold improvements, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh\u0026ndash;j). Flow cytometric observations demonstrated that immune responses stimulated by NIR-PIT alone or ICB alone were limited, whereas NIR-PIT with PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e significantly activated DC maturation and recruited T cells to exert anti-tumor effects.\u003c/p\u003e \u003cp\u003eFurthermore, NIR-PIT of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e significantly elevates the levels of cytokines IL-4, TNF-α, IFN-γ, and IL-6 \u003cem\u003ein vivo\u003c/em\u003e. Compared to the control group, the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group exhibited a 2.38-fold increase in IL-4 levels (2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32), a 4.35-fold increase in IFN-γ levels (3.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 vs. 0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22), a 2.35-fold increase in IL-6 levels (2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19), and a 1.80-fold increase in TNF-α levels (159.68\u0026thinsp;\u0026plusmn;\u0026thinsp;23.56 vs. 88.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99). Furthermore, the aPD-L1 group showed substantially elevated cytokine levels: IL-4 increased by 30.95-fold (30.95\u0026thinsp;\u0026plusmn;\u0026thinsp;9.39 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32), IFN-γ increased by 19.82-fold (116.64\u0026thinsp;\u0026plusmn;\u0026thinsp;8.05 vs. 0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22), IL-6 increased by 11.83-fold (11.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19), and TNF-α increased by 7.32-fold (649.39\u0026thinsp;\u0026plusmn;\u0026thinsp;116.81 vs. 88.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99). Elevated levels of IL-6, IL-4, IFN-γ, and TNF-α are known to induce cytokine storms, with IL-6 serving as a key initial driver in this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek\u0026ndash;n).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of combination therapy on the systemic immunological response\u003c/h3\u003e\n\u003cp\u003eTo assess the anti-tumor efficacy of combination therapy on systemic immunological responses, mouse models with bilateral tumors were used. Once the primary tumor reached approximately 50 mm\u0026sup3; in volume, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e was intravenously administered via the tail vein. At 24 h post-injection, the primary tumors were exposed to NIR irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe results demonstrated that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR treatment significantly inhibited tumor growth at both the primary and distant sites, compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u0026ndash;f). In the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group, substantial therapeutic effects were observed on primary tumors, with significant inhibition. Distant tumors also exhibited a notable therapeutic response, indicating that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR had a systemic inhibitory effect. The inhibitory effect at distant tumors began to emerge 4 d post-treatment, indicating that systemic immune activation takes longer to develop than the immediate direct NIR-PIT effects observed in primary tumors. Furthermore, the group treated with PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e alone showed moderate tumor inhibition compared with the control group, suggesting that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e has inherent anti-tumor properties due to its ICB capabilities. Based on this hypothesis, we analyzed immune cells within the TME of mice, focusing on T cells.\u003c/p\u003e \u003cp\u003eThe results showed significant immune activation of the TME in the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group. The proportions of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells were markedly higher than those in the control group, with CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells increasing approximately 16-fold and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells increasing about 3.5-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg, i\u0026ndash;j). In contrast, the proportion of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e regulatory T cells (Tregs) decreased by approximately 6.7-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh, k), indicating that PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR activated the immune environment and alleviated immune suppression. This comprehensive evaluation highlighted the functionality of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR therapy in targeting both primary and distant tumors. The NIR-PIT with PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e not only achieved immediate in situ tumor control but also promoted a systemic anti-tumor immune response, providing a promising approach for treating metastatic cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eActivation of the HSP70/AKT/mTOR axis via NIR-PIT induces tumor recurrence\u003c/h3\u003e\n\u003cp\u003eAlthough NIR-PIT has shown effective therapeutic outcomes, tumor recurrence remains a significant challenge, with some tumors exhibiting resistance to NIR-PIT (Supplementary Fig.\u0026nbsp;15). To further understand the mechanism of tumor resistance, we conducted transcriptomic sequencing of recurrent tumors from the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group as well as tumors from the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e and PBS control groups. Our analysis revealed that 20,700 genes were detected in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Volcano plots indicated that, in the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group, compared to the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e group, 2,888 genes were upregulated, while 2,099 genes were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Additionally, compared with the PBS group, the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group had 2,657 upregulated genes and 1,729 downregulated genes, respectively (Supplementary Fig.\u0026nbsp;16). To identify the pathways potentially driving tumor recurrence, we performed a KEGG pathway analysis, which highlighted the significant upregulation of the PI3K/AKT/mTOR pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, d). The mTOR pathway is crucial for promoting cell proliferation which is linked to tumor recurrence.\u003c/p\u003e \u003cp\u003ePrevious research has indicated that heat shock protein 70 (HSP70) activates the mTOR pathway\u003csup\u003e49\u003c/sup\u003e. Given that NIR-PIT acts as a stressor that induces the expression of HSPs, including HSP70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee, f), we proposed that NIR-PIT-induced HSP70 expression upregulates the PI3K/AKT/mTOR pathway, thereby promoting tumor cell proliferation. To validate this hypothesis, we measured HSP70 expression in tumor tissues following NIR-PIT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef), observing a significant increase in HSP70 protein levels. Subsequently, we knocked down HSP70 in CT26 cells and examined the PI3K/AKT/mTOR pathway, and observed a reduction in its activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg). Therefore, we conclude that the activation of the PI3K/AKT/mTOR pathway is driven by the upregulation of HSP70, leading to NIR-PIT resistance and tumor recurrence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNIR-PIT combined with mTOR inhibitor rapamycin can reduce tumor recurrence\u003c/h2\u003e \u003cp\u003eNIR-PIT can activate the immune response; however, it may also trigger cellular stress, elevate HSP70 levels, and activate the PI3K/AKT/mTOR signaling pathway, potentially resulting in therapeutic resistance in tumor cells. To address this issue, a specific mTOR inhibitor and FDA-approved drug, rapamycin, was integrated into this study to suppress the PI3K/AKT/mTOR pathway, thereby enhancing the effects of NIR-PIT and reducing recurrence rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). In the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR group, 6 out of 20 mice experienced recurrence, yielding a recurrence rate of 33%. In contrast, no recurrence was observed in the 20 mice treated with PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR combined with rapamycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb, c). To better replicate clinical conditions, patient-derived clinical samples from breast cancer patients were used to establish an \u003cem\u003ein situ\u003c/em\u003e murine PDX model, demonstrating significant therapeutic outcomes and exceptionally high safety in the combined treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed, e and Supplementary Fig.\u0026nbsp;17). Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef presents \u003cem\u003eex vivo\u003c/em\u003e tumor images from the two PDX model groups, with significant differences in tumor weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eg). These results confirmed the enhanced efficacy of this combination treatment, with significantly reduced tumor recurrence and fewer side effects. In general, NIR-PIT therapy elevates HSP70 expression, triggering the activation of the PI3K/AKT/mTOR pathway and leading to tumor recurrence. Rapamycin selectively inhibits mTOR, effectively suppressing PI3K/AKT/mTOR pathway activation, tumor recurrence, and enhancing treatment outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eClinical cancer immunotherapy faces several challenges, including low immune response rates and limited efficacy, particularly in treating \u0026ldquo;cold\u0026rdquo; tumors. To address these issues, we developed a novel carrier-free, self-assembled NIR-PIT nanodelivery system, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, which integrates tumor-targeted delivery, immune checkpoint blockade and photothermal function while minimizing toxicity. The PD-L1 targeting immune-peptide PSP specifically binds to PD-L1 expressed on tumor cell surfaces, inhibiting tumor immune evasion by blocking the PD-1/PD-L1 pathway. Meanwhile, IR-CF\u003csub\u003e3\u003c/sub\u003e acting as a photothermal response component converts light energy into heat under NIR irradiation, activating the TME and enhancing immune responses. Unlike traditional external carrier-based delivery systems, the self-assembled, carrier-free PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NFs avoid issues related to nanocarrier safety, uncontrolled drug release, and inefficient targeting. This self-assembly structure improves drug biodistribution and targeting to the tumor site and extends the drug\u0026rsquo;s half-life and circulation time, reducing systemic exposure and minimizing side effects. Furthermore, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e enables multifaceted therapeutic effects with a single dose, increasing the combined immunotherapeutic effects with reduced toxicity, simplified treatment, and improved clinical feasibility.\u003c/p\u003e\n\u003cp\u003eIn terms of therapeutic efficacy, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e plus NIR outperforms traditional PD-L1 antibody immunotherapy. The mild photothermal therapy activates the immune microenvironment, enhances tumor tissue permeability, reduces damage to normal tissues, and promotes synergistic effects between immunotherapy and photothermal therapy\u003ca href=\"#_ENREF_50\" title=\"Saeed, 2019 #49\"\u003e\u003csup\u003e50\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_51\" title=\"Stapleton, 2018 #50\"\u003e\u003csup\u003e51\u003c/sup\u003e\u003c/a\u003e. PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e plus NIR effectively induces the ICD effect, which not only directly damages tumor cells but also releases a range of immune-stimulatory molecules, such as HMGB1. These released molecules promote the activation of DCs, enhancing their antigen-presenting capacity, which induces the activation and proliferation of tumor-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells, improves the TME by increasing immune cell infiltration, and significantly boosts immunotherapeutic outcomes\u003ca href=\"#_ENREF_50\" title=\"Saeed, 2019 #49\"\u003e\u003csup\u003e50\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_52\" title=\"Tang, 2021 #47\"\u003e\u003csup\u003e52\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_53\" title=\"Zhao, 2024 #48\"\u003e\u003csup\u003e53\u003c/sup\u003e\u003c/a\u003e. More importantly, unlike traditional immunotherapies, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e also effectively reduces the risk of cytokine storms, particularly by lowering levels of pro-inflammatory cytokines such as IL-6, IFN-\u0026gamma;, and TNF-\u0026alpha; etc. Furthermore, it minimizes the severe side effects commonly associated with potent immunotherapies, thereby enhancing treatment safety\u003ca href=\"#_ENREF_54\" title=\"Fajgenbaum, 2020 #45\"\u003e\u003csup\u003e54\u003c/sup\u003e\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite these promising results, NIR-PIT still encounters resistance in certain recurrent tumors, which may limit its long-term treatment efficacy. Our transcriptomic analysis identified the HSP70/AKT/mTOR pathway as a key mediator of this resistance. And \u0026nbsp;we further combined PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e with rapamycin, a selective mTOR pathway inhibitor, to overcome this challenge\u003ca href=\"#_ENREF_55\" title=\"Joshi, 2018 #51\"\u003e\u003csup\u003e55\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_56\" title=\"Goloudina, 2012 #52\"\u003e\u003csup\u003e56\u003c/sup\u003e\u003c/a\u003e. Rapamycin alleviates NIR-PIT-induced resistance by inhibiting the mTOR pathway, thereby reducing the risk of tumor recurrence. This combination strategy takes advantage of the synergistic effects of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e and rapamycin, significantly improving long-term therapeutic efficacy and prolonging treatment benefits.\u003c/p\u003e\n\u003cp\u003eIn summary, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e offers great opportunity for clinical translation due to its simple self-assembled NF structure, which significantly enhances therapeutic efficacy while reducing systemic toxicity. Its ability to deliver multi-faceted therapeutic effects with a single dose simplifies treatment regimens, improving patient compliance and efficacy. The combination with rapamycin, an FDA-approved drug, further strengthens its clinical feasibility, leveraging rapamycin\u0026rsquo;s established safety and efficacy. This approach boosts therapeutic outcomes and reduces resistance, providing a promising and effective strategy for treating \u0026ldquo;cold tumors\u0026rdquo; and advancing cancer immunotherapy.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\n\u003ch3\u003eMaterials and reagents\u003c/h3\u003e\n\u003cp\u003ePeptide NQ40 (\u0026ldquo;NTKYFYEDQ\u0026rdquo;) was synthesized by Nanjing TAIYE Co. Ltd. (Nanjing, China). InVivo MAb anti-mouse PD-L1 (B7-H1) was obtained from BioX (#BE0101; Lebanon, NH, USA). The primary antibodies used include anti-calreticulin rabbit pAb (GB112134), anti-HMGB1 rabbit pAb (GB11103), and anti-HSP70 rabbit pAb (GB11241), all sourced from Servicebio (Wuhan, China). The secondary antibodies, Cy3-conjugated goat anti-rabbit IgG (GB21303) and Alexa Fluor\u0026reg; 488-conjugated goat anti-rabbit IgG (GB25303), were also obtained from Servicebio. Fluorochrome-conjugated anti-mouse monoclonal antibodies, including BB515 rat anti-mouse CD45 (564590; BD, Franklin Lakes, NJ, USA), APC/Fire\u0026trade; 750 anti-mouse CD3 (100248; BioLegend, San Diego, CA, USA), APC anti-mouse CD4 (100412; BioLegend), PE/Cyanine7 anti-mouse CD8a (100722; BioLegend), APC/Cyanine7 anti-mouse CD11c (117324; BioLegend), PE anti-mouse CD80 (104708; BioLegend), Brilliant Violet 421\u0026trade; anti-mouse CD86 (105032; BioLegend), APC anti-mouse I-A/I-E (107614; BioLegend), PE anti-mouse/human CD11b (101208; BioLegend), PE/Cyanine7 anti-mouse Ly-6G/Ly-6C (Gr-1) (108416;BioLegend), PE anti-mouse CD25 (102007; BioLegend), FOXP3 Monoclonal Antibody (FJK-16s), PerCP-Cyanine5.5 (45-5773-82; Thermo Fisher Scientific, Waltham, MA, USA), PE/Cyanine7 anti-mouse/human CD44 (103030; BioLegend), APC anti-mouse CD62L (104412; BioLegend), PE anti-mouse CD4 (116005; BioLegend), TruStain FcX\u0026trade; (anti-mouse CD16/32) Antibody (101320; BioLegend).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eFirst, Solution A was prepared by dissolving PSP (2 mg/mL) in PBS (pH 7.4), while Solution B was made by dissolving IR-CF\u003csub\u003e3\u003c/sub\u003e in dimethyl sulfoxide (20 mg/mL). Solution A (2 mL) was transferred to a 300 W ultrasonic homogenizer, before 100 \u0026micro;L of Solution B was subjected to ultrasound treatment. The mixture was sonicated for 2 h using an ultrasonic homogenizer. The mixed solution was then removed and placed in a dialysis bag with a 1000 Da cutoff. After 12 h of dialysis in PBS solution, the liquid was removed from the dialysis membrane and stored in a 4\u0026deg;C refrigerator for incubation. After 24 h, the NFs were stably assembled.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eCT26-Luc cells (luciferase-expressing mouse colorectal cancer cells) were sourced from the Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences (Beijing, China). CT26-Luc cells were maintained in a standard medium with 10% fetal bovine serum (FBS; Gibco, Invitrogen) and 100\u0026times; penicillin\u0026ndash;streptomycin solution (Solarbio, Beijing, China). The CT26-Luc cells were incubated at 37\u0026deg;C with 21% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eBALB/c mice and NTG mice (NOD-scid IL2Rγ \u0026minus;/\u0026minus;), both 6 weeks old, were obtained from SPF Biotechnology Co. Ltd. (Beijing, China). Animals were kept in a specific pathogen-free (SPF) environment at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C with a 12-hour light/dark cycle and free access to food and water. Animal procedures followed the guidelines for ethical research use set by Peking Union Medical College Hospital (permit number: XHDW-2022-016).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of subcutaneous mouse model\u003c/h2\u003e \u003cp\u003eBALB/c mice were injected in their flanks with 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CT26-Luc cells per mouse to generate the subcutaneous tumor model. \u003cem\u003eIn vivo\u003c/em\u003e photothermal immunotherapy was performed on CT26 tumor-bearing mice, which were randomly allocated to seven groups: (i) PBS, (ii) IR-CF\u003csub\u003e3\u003c/sub\u003e (675 \u0026micro;g/mL, i.v.), (iii) aPD-L1 (2 mg/mL, i.v.), (iv) PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e (675 \u0026micro;g/mL, i.v.), (v) NIR (1 W/cm\u003csup\u003e2\u003c/sup\u003e, 10 min), (vi) CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR, (vii) PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR. Bilateral CT26 tumor-bearing mice received treatment with PBS or PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, with or without NIR, as described previously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of PDX model\u003c/h2\u003e \u003cp\u003eFresh human breast tumor fragments were transplanted \u003cem\u003ein situ\u003c/em\u003e into the right mammary glands of anesthetized NTG mice. The mice were then observed in SPF conditions. Tumor growth was measured two-dimensionally using calipers. The formula for calculating the tumor volume (TV) was TV = (width\u003csup\u003e2\u003c/sup\u003e x length) \u0026times; 0.5. The tumors were typically passaged when the TV reached 1 cm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunogenic cell death expression assays\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was used to investigate CRT overexpression. The CT26-Luc cells were treated with PBS, CF\u003csub\u003e3\u003c/sub\u003e, and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e for 12 h, respectively. The cells were exposed to an 808 nm laser (1 W/cm\u003csup\u003e2\u003c/sup\u003e, 10 min) and then incubated for an additional 24 h. Subsequently, the cells were washed with PBS, fixed using 4% formaldehyde, and treated with 3% bovine serum albumin. The rabbit anti-CRT antibody at a 1:100 dilution was applied and left to incubate overnight at 4\u0026deg;C. After three washes with phosphate buffered saline with Tween-20 (PBST), the cells were stained with a Cy3-conjugated goat anti-rabbit secondary antibody at a 1:300 dilution, and DAPI was used to stain the nuclei. The fluorescence microscope (Nikon, Japan) was used to measure the expression level of CRT.\u003c/p\u003e \u003cp\u003eOverexpression of HMGB1 was investigated with an enzyme-linked immunosorbent assay (ELISA) kit (SEA399Mu 96T, Cloud-clone, Wuhan, China). The CT26-Luc cells were treated with PBS, IR-CF\u003csub\u003e3,\u003c/sub\u003e and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e for 12 h, respectively. The cells were exposed to an 808 nm laser (1 W/cm\u003csup\u003e2\u003c/sup\u003e, 10 min) and then incubated for an additional 24 h. The supernatant was spun at 4\u0026deg;C for 15 min at 3000 RPM and used immediately for the experiment. HMGB1 release was measured using an HMGB1 ELISA kit following the provided instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003efluorescence molecular imaging (FMI)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCT26 tumor-bearing mice were allocated into three groups. In the blocking group, 200 \u0026micro;g of aPD-L1 was administered intraperitoneally 12 h before imaging, and again 1 h before imaging. Mice anesthetized with isoflurane were subjected to FMI following intravenous administration of IR-808, with or without PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, on the IVIS Spectrum system (Perkin Elmer). Images were acquired before injection and at subsequent time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 48 h), using excitation and emission wavelengths of 745 and 800 nm, respectively. 2 d after injection, the tumor-bearing mice from each group were euthanized to retrieve the major organs (heart, liver, spleen, lung, kidney, and tumor) for imaging of the excised tissues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ebioluminescence imaging (BLI)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBilateral CT26 tumor-bearing mice were monitored using BLI. Mice were anesthetized with 1.5% isoflurane in oxygen and intraperitoneally injected with D-luciferin (150 \u0026micro;g/g body weight; Perkin Elmer) 8 min before imaging. BLI images were acquired with the IVIS Spectrum system to monitor tumor signals at different time points after treatment in each group (pre, 4, 8, 12, and 16 d).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ephotothermal conversion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCT26 tumor-bearing mice were allocated to three groups (PBS, IR-CF\u003csub\u003e3,\u003c/sub\u003e and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e) according to the average tumor volume and injected intravenously 100 \u0026micro;L of IR-CF\u003csub\u003e3\u003c/sub\u003e and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e at the same concentration of 675 \u0026micro;g/mL. Tumors that were subjected to localized injections of PBS were used as a control group. All tumors were then irradiated with an 808 nm laser (1.0 W/cm\u003csup\u003e2\u003c/sup\u003e, 10min). During irradiation, the IR images of the mice were obtained with a compact thermal imaging camera (FLIR E60). After treatment, the data including tumor volumes and body weights were collected at predetermined time intervals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEvaluating anti-tumor efficacy in the CT26 tumor model\u003c/h2\u003e \u003cp\u003eThe anti-tumor effects of NFs were evaluated in CT26 tumor-bearing mice. Once the tumor volumes reached around 80 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly allocated into seven groups: PBS (control), IR-CF\u003csub\u003e3\u003c/sub\u003e, aPD-L1, PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e, NIR, IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR, and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR. Mice in the IR-CF\u003csub\u003e3\u003c/sub\u003e and IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR groups received an intravenous injection of 100 \u0026micro;L IR-CF\u003csub\u003e3\u003c/sub\u003e (675 \u0026micro;g/mL). Mice in the PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e and PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR groups were injected intravenously with 100 \u0026micro;L PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e (IR-CF\u003csub\u003e3\u003c/sub\u003e: 675 \u0026micro;g/mL) per mouse. NIR irradiation (1.0 W/cm\u003csup\u003e2\u003c/sup\u003e, 10 min) was performed 24 h after injection. Mice in the aPD-L1 groups received an intravenous injection of 100 \u0026micro;L aPD-L1 (2 mg/mL). Two methods were employed to determine tumor volume. One method used the formula (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/2, whereas the other utilized the bioluminescence intensity determined by the imaging system mentioned above. ICD expression following treatment was assessed by analyzing cleaved caspase-3, CRT, and HMGB1 levels in tumor regions using immunohistochemistry and immunofluorescence.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eanalysis of T cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo perform immune cell analysis by flow cytometry, tumors or spleens from mice subjected to different treatments were harvested and stained in accordance with the provided protocols. In short, tumor cells were blocked with CD16/32 antibody (101320; BioLegend) before being stained with the following antibody combinations: CTLs and Th cells (anti-CD45-BB515, anti-CD3-APC-Fire750, anti-CD4-APC, and anti-CD8-PE-Cy7); DCs (anti-CD45-BB515, anti-CD11c-APC-Cy7, anti-CD80-PE, anti-CD86-BV421, and anti-MHCII-APC); Treg (anti-CD45-BB515, anti-CD4-APC, anti-CD25-PE, and anti-FOXP3-PerCP-Cy5.5). Finally, monoclonal antibody-stained cell suspensions were analyzed using a flow cytometer (NL-CLC3000; Cytek, Fremont, CA, USA) in accordance with standard protocols. For each test, 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCytokine detection\u003c/h2\u003e \u003cp\u003eTNF-α, IL-4, IFN-γ, and IL-6 (all from Multisciences Biotech, Hangzhou, China) in mouse serum were measured with ELISA kits following the standard protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism V10.1.2 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis. Results are shown as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean or standard deviation, as indicated. To compare two groups, Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests were applied (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was deemed statistically significant).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Beijing Natural Science Foundation (Grants No. 7254548, 7252292, 7244524), the National Natural Science Foundation of China (Grants No. U24A20731, 81227901, 81930053, 62027901, 32101153), Young Elite Scientists Sponsorship Program by BATSA (BYESS2023244), Emerging Engineering Interdisciplinary-Young Scholars Project (PKU2024XGK007), Peking University, and Peking University Medicine Sailing Program for Young Scholars\u0026apos; Scientific \u0026amp; Technological Innovation (BMU2024YFJHPY015), the Fundamental Research Funds for the Central Universities.\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to Prof. Weizhi Wang from the School of Chemistry and Chemical Engineering at Beijing Institute of Technology for providing the peptide sequence, Prof. Aiguo Wu from the Ningbo Institute of Materials Technology \u0026amp; Engineering for synthesizing IR-CF\u003csub\u003e3\u003c/sub\u003e, and the Analysis \u0026amp; Testing Center at Beijing Institute of Technology.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eY. D., H. X., W. H., and Z. J. conceptualized and supervised the entire research project. X. Z. authored the manuscript. Both X. Z. and Y. D. engineered and executed the imaging, therapeutic efficacy, and mechanistic studies. Z. J. and X. T. were responsible for synthesizing the materials and their characterization. W. Z. conducted in vivo imaging experiments on small animals. J. W. assisted in the collection of samples from these animals. M. S. contributed to establishing and maintaining the animal models. H. G. and C. L. participated in data analysis and further characterization of properties.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interest.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and the Supplementary information or available from the author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGalon, J. \u0026amp; Bruni, D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. \u003cem\u003eNat. Rev. Drug. Discov.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 197-218 (2019).\u003c/li\u003e\n \u003cli\u003eKhosravi, G. R.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Immunologic tumor microenvironment modulators for turning cold tumors hot. \u003cem\u003eCancer. 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Lett.\u003c/em\u003e \u003cstrong\u003e325\u003c/strong\u003e, 117-124 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5827209/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5827209/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough immune checkpoint blockade (ICB) therapies have shown clinical benefit, ICB remains limited effect on most \u0026ldquo;immune-cold\u0026rdquo; solid tumors, i.e., tumors with low immune infiltration. Near-infrared photoimmunotherapy (NIR-PIT) converts \u0026ldquo;cold\u0026rdquo; tumors into \u0026ldquo;hot\u0026rdquo; tumors, thereby enhancing immune responses and improving ICB efficacy. Hence, developing a strategy that can integrate NIR-PIT and ICB treatment is desirable. In this study, we designed a unique carrier-free PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e nanofiber (NF), self-assembled from the PD-L1 targeting immune-peptide NTGYFYGDQ (PSP) and NIR-PIT agents (IR-CF\u003csub\u003e3\u003c/sub\u003e). The NFs enable precise tumor targeting and immune checkpoint inhibition by specifically binding to PD-L1 on the tumor cell surface, also providing an NIR-mediated photothermal effect. Applying PSP@IR-CF\u003csub\u003e3\u003c/sub\u003e NFs with NIR induced mild NIR-PIT, which effectively activated the tumor immune microenvironment and treat tumors with lower immunotoxicity. Moreover, we identified that the HSP70/AKT/mTOR signaling pathway, which regulates tumor resistance and recurrence, was activated after PIT. By incorporating mTOR inhibitors like rapamycin, the combination treatment can reduce tumor resistance to NIR-PIT and decrease recurrence, thereby significantly improving therapeutic outcomes. This innovative combination therapy has the potential to revolutionize \u0026ldquo;cold\u0026rdquo; tumor treatment by offering more precise interventions that markedly enhance immunotherapeutic efficacy, reduce toxicity, and improve patient outcomes.\u003c/p\u003e","manuscriptTitle":"Combined immune-peptide nanofiber with HSP70/AKT/mTOR axis blockade enhances near-infrared photoimmunotherapy, inhibiting tumor growth and recurrence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 10:29:34","doi":"10.21203/rs.3.rs-5827209/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd99fad9-7475-4681-b28d-0c52173f742c","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43370251,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"},{"id":43370252,"name":"Biological sciences/Biological techniques/Nanobiotechnology"}],"tags":[],"updatedAt":"2025-08-21T12:17:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-28 10:29:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5827209","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5827209","identity":"rs-5827209","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}