Near‐infrared laser‐activated aggregation‐induced emission nanoparticles boost tumor carbonyl stress and immunotherapy of breast cancer

The induction of tumor carbonyl stress is reported to efficiently revert immune suppression in the tumor microenvironment and enhance cancer immunotherapy. However, low oxygen concentration due to inherent tumor hypoxia limits its catalytic effect. Herein, an injectable thermosensitive hydrogel system (named APH) is developed for co‐loading of near‐infrared (NIR) aggregation‐induced emission (AIE) nanoparticles and plasma amine oxidase (PAO) for boosting carbonyl stress and enhancing antitumor immunity. Upon 808 nm NIR laser irradiation, the AIE nanoparticles trigger a mild‐temperature (around 45°C) photothermal effect in the tumor site, which significantly relieves tumor hypoxia and promotes the catalytic effect of released PAO to inhibit the growth of Myeloid‐derived suppressor cells. Remarkably, the synergistic therapeutic effect of APH is verified through a significant inhibitory effect on the distant tumor, enhanced immune memory, and effective suppression of postoperative recurrence, rechallenge, and metastasis. Overall, the combined effect of AIE‐mediated photothermal therapy and carbonyl stress by APH upon NIR irradiation therapy can significantly activate cancer immunotherapy, making it a promising treatment approach for cancer treatment.

Polyamines are a kind of low molecular weight, polycationic, aliphatic nitrogen-containing substances, mainly including spermine, spermidine, and putrescine.The homeostasis of polyamines is crucial for maintaining normal cell growth and proliferation in the body. [9]Abnormal changes in polyamine metabolism often occur in tumor cells. [10]roteins expressed by oncogenes such as MYC, RAS, and BRAF V600E maintain high levels of polyamine pools in tumor microenvironment by inducing polyamine transport systems to increase polyamine transport, reduce polyamine breakdown, and upregulate polyamine biosynthesis. [11,12]ccumulating studies show that polyamines have antiinflammatory and immunosuppressive properties, and clearing the tumor site of polyamines or inhibiting their synthesis can effectively improve the effectiveness of immunotherapy. [13]Fortunately, polyamine oxidase (PO) regulates the degradation of polyamines.Polyamines can be metabolized by PO to produce reactive oxygen species (ROS) including hydrogen peroxide (H 2 O 2 ) and cytotoxic aldehydes including acrolein by enzyme catalysis reaction (Polyamine + O 2 + H 2 O PO → H 2 O 2 + acrolein + NH 3 ). [14]crolein is one of the reactive carbonyl species that has high electrophilicity activity and can covalently bind to nucleophilic sites such as proteins, nucleic acids, and phospholipids through Michael addition or Schiff base reactions, leading to cytotoxicity. [15]Therefore, PO has great potential for enhancing tumor immunotherapy.[18][19][20] The negative feedback may further promote the hypoxia environment, making cancer cells more resistant to traditional therapy. [21]herefore, another efficient therapeutic method is urgently needed to promote PO-mediated carbonyl stress and realize synergistic outcomes for stimulating immunotherapy of tumors.
It is reported that mild photothermal therapy (PTT), offering a moderate heating effect to around 45 • C, can accelerate blood circulation and relieve hypoxia, [22][23][24] showing potential for relieving hypoxia.[32] The active intramolecular motion of AIE luminaires (AIEgens), combined with molecular rotors, can provide efficient non-radiative decay to release the excited laser energy as heat, enabling the production of photothermal materials.In view of the above considerations, mild PTT based on AIEgens could remedy the defects of PO-mediated therapy and serve as an ideal way to enhance the catalytic ability of PO. [33][34][35] Herein, we designed an injectable thermosensitive hydrogel system called APH by co-loaded with a near-infrared (NIR)-absorbing AIE molecule namely 2TT-oC26B and plasma amine oxidase (PAO, a kind of PO) for synergistically boosting carbonyl stress for enhancing anti-tumor immunity.By encapsulating 2TT-oC26B to nanoparticles, the fabricated AIE nanoparticles (NPs) have strong absorption in the NIR region and can rapidly heat up under 808 nm laser irradiation, thus exerting cancer PTT with high biocompatibility. [36]n the other hand, agarose hydrogel was chosen because of the ability of light-controlled drug release, and stable retention into the tumor site even after a single injection. [37,38]fter injecting APH into the tumor, 808 nm NIR laser irradiation was performed, and the tumor site was heated to around 45 • C. At this time, we found that the hypoxia situation at the tumor site was significantly improved.The results of bilateral tumor experiments in vivo showed that after APH plus NIR treatment, the polyamine content and myeloid-derived suppressor cells (MDSCs) in the tumor site decreased significantly.The number of mature dendritic cells (DC) in mouse lymph nodes and the CD8 + T lymphocytes in the distal tumor increased, which resulted in a significant inhibitory effect on the distal tumor.In addition, we further found that the number of central memory T cells in the peripheral blood increased after APH treatment upon NIR irradiation.Compared with the control group, the recurrence and rechallenge tumor were inhibited after APH treatment.Overall, APH can significantly activate the body's tumor immune system, making it a promising treatment approach for cancer treatment.

Preparation and characterization of APH
2TT-oC26B was prepared and characterized according to previous work (Scheme S1 and Figure S1).As shown in Figure 1A, the resulting molecule exhibits twisted molecular rotors and a deformed backbone, which can prevent close packing and energy dissipation in the aggregate form.The 2TT-oC26B molecule's photoluminescence spectra and AIE curves in dimethyl sulfoxide (DMSO)/water mixtures with various water fractions demonstrate that the molecule was weakly emissive in DMSO solution due to the active intramolecular motion but showed intense emission at NIR-II region in aggregates because of restriction of intramolecular motion, displaying a typical AIE property (Figure 1B,C).To enhance the biocompatibility and water solubility, DSPE-Polyethylene glycol was used to encapsulate the hydrophobic 2TT-oC26B and fabricated as the AIE NPs.The synthesized AIE NPs were uniform in size with an average particle size of ∼100 nm according to transmission electron microscopy and dynamic light scattering analyses (Figure 1D).Furthermore, 2TT-oC26B exhibited good stability at room temperature over 7 days (Figure S2).Subsequently, we investigated the photophysical properties of AIE nanoparticles.2TT-oC26B displayed a typical NIR-I absorption at 690 nm and NIR-II emission at 1014 nm (Figure 1E).The strong absorption of 2TT-oC26B in the NIR-I region encouraged us to further investigate its photothermal conversion effects under 808 nm laser irradiation.As shown in Figure 1F, 2TT-oC26B underwent steady and similar temperature alterations under laser irradiation during the four heating and cooling cycles.Moreover, 2TT-oC26B exhibited much higher photostability compared with indocyanine green, a conventional photothermal reagent.The AIE NPs and PAO were blended into thermosensitive agarose hydrogel to fabricate the synergistic therapeutic system, namely APH.The three-dimensional morphology of APH was confirmed by scanning electron microscopy.The interstitial structure inside the hydrogel is suitable for loading molecular drugs (Figure 1G).Rheology measurement of APH showed the reduction of the storage modulus at an increasing temperature under laser irradiation, indicating the hydrogel softening process and potential drug release power (Figure 1H).The temperature elevation of different 2TT-oC26B concentrations (0, 50, 100, and 200 μg/mL) under laser irradiation was further evaluated (Figure 1I).The result demonstrated that the temperature change of APH is proportional to the concentration of AIE NPs, showing the controllability of photothermal conversion efficiency.In addition, infrared thermal images before and after laser irradiation also verified the good photothermal performance of APH Figure 1J, which also indicated that 2TT-oC26B NPs encapsulated in APH could provide sufficient high temperature to soften agarose from the gel state into the solution state, facilitating the release of PAO for subsequent therapeutic evaluation.PAO release was further quantified by BCA Protein Assay Kit after fourtime laser irradiation in Figure 1K.To testify to the catalytic power of PAO, the generation of H 2 O 2 by APH, containing 20 μg/mL PAO and different concentrations of spermine, was determined by ultraviolet-visible absorbance of chromogenic tetramethylbenzidine (TMB) in the presence of horseradish peroxidase.The generated H 2 O 2 concentration gradually increased with the additional reaction time and spermine concentration, indicating that polyamines can be catalyzed by APH with time dependence and dose dependence (Figure 1L).Overall, APH was successfully prepared to display excellent photothermal properties and controlled drug release power.In addition, the released PAO from APH can effectively catalyze spermine to generate H 2 O 2 , showing synergistic therapeutic potential for cancer.

In vitro anti-cancer effect of APH
Considering the excellent photophysical properties of the APH system, we then explored the in vitro killing effect of APH on 4T1 breast cancer cells.4T1 cells were pre-incubated with 350 μM spermine and treated with 5 different groups: (1) PBS+NIR (808 nm, 0.5 W/cm  2A,B, the contents of acrolein in PAO and APH+NIR groups were significantly higher than those in other groups under normoxic conditions, while the acrolein production under hypoxic conditions was lower than that in the corresponding group under normoxic conditions.This indicated that PAO released by APH under laser irradiation could effectively catalyze spermine to produce acrolein, and the catalytic action was limited by oxygen concentration.Subsequently, the DCFH-DA fluorescent probe was used to further detect ROS levels in 4T1 cells after different treatments.As shown in Figure 2C,D and Figure S3, the strongest fluorescence could be detected in PAO and APH+NIR groups, confirming that the production of intracellular H 2 O 2 and ROS was related to the catalytic nature of PAO.Based on the excellent ability of APH+NIR to produce H 2 O 2 and acrolein, the cytotoxic effects of different treatments on 4T1 cells in the presence of sperminine were evaluated by MTT assay.As shown in Figure 2E, the APH+NIR group exhibited stronger killing effects than AH+NIR and PAO.Furthermore, the combination index of the two drugs was calculated to be 0.53 according to the equation by Chou-Talalay Method, which quantitatively depicts the high synergistic effect of PAO and AIE NPs in the APH gel. [39,40]In addition, the experimental results shown in Figure 2F indicated that the cytotoxic effects

In vitro induction of immunogenic cell death
Next, we investigated whether the synergistic therapeutic effect of APH could evoke the immunogenic cell death (ICD) effect on 4T1 cells.ICD is an important means of inducing tumor immunotherapy, which can cause surface-exposed calreticulin (CRT) and stimulate exocrine damage-associated molecular patterns (DAMPs), including high mobility group Box 1 (HMGB1) or adenosine triphosphate (ATP).CRT expressions, HMGB1 release, and ATP secretion on the cell surface of APH-treated cells were then analyzed.CRT can be used as an effective phagocytosis signal for tumor cells, which can cause tumor cells to be phagocytosed by binding to related protein receptors on the surface of phagocytes, thus causing tumor immune response in the body.As shown in Figures 3A,C, AIE NPs mediated-PTT (AH+NIR group) slightly interfered with CRT expression in treated cells compared to PBS and APH groups, while the PAO group showed moderate CRT exposure.The cells treated with APH could significantly promote the expression of CRT under light irradiation, exhibiting a 5-time higher CRT exposure level than that in the PBS control group.Furthermore, other DAMPs of ICD including HMGB1 and ATP were also investigated.As shown in Figure 3B,D,E, APH+NIR-treated cells produced the highest level of released HMGB1 and secreted ATP under hypoxic conditions, and this effect was more significant under normoxic conditions.These results suggested that APH-mediated PTT in combination with carbonyl stress can induce effective ICDs in cancer cells, which benefits tumor immunotherapy.

In vivo antitumor study
Encouraged by the above results, we next evaluated the in vivo therapeutic efficacy of APH.In order to explore the in vivo metabolism of nano-drugs, NIR fluorescence images of BALB/c mice carrying 4T1 tumors were used to assess the biological distribution and pharmacokinetics after intratumoral injection of APH.The content of 2TT-oC26B in plasma after intratumoral injection is shown in Figure S4.Due to the continuous release of 2TT-oC26B in the hydrogel, it showed a trend of first increasing and then being metabolized.As shown in Figure S5, tumor tissues of 4T1 tumor-bearing mice showed obvious fluorescence signals at 1 h post-injection.The fluorescence intensity at the tumor site decreased over time, which may be related to the efficient clearance of APH from the body.We evaluated whether APH could effectively relieve hypoxic conditions to explore the intratumoral behaviors of the systemically administered particles (Figure 4B,C).Anti-pimonidazole antibody (green) and DAPI (blue) were used to stain hypoxic regions and the cell nuclei, respectively.We found that the PBS-injection tissue contained abundant green fluorescence accounting for 79.83 ± 3.91% of the whole tumor tissue, indicative of obvious hypoxia status.Notably, the AH+NIR and the APH+NIR groups substantially decreased the hypoxic areas, with a nearly 3.5-fold decline in comparison with the PBS group.It is proposed that 2TT-oC26B-mediated mild PTT can relieve the anoxic situation in the tumor site, which will be conducive to our subsequent treatment experiments.Next, we explored the in vivo antitumor efficacy of APH.As shown in Figure 4A S6).APH treatment only slightly delayed the tumor growth, which may be related to the slow release of PAO from the hydrogel in the tumor site.PAO treatment group and AH+NIR group had a more significant inhibitory effect on the growth of both primary and distal tumors.Studies have shown that PAO can rapidly deplete polyamines at tumor sites, producing large amounts of highly toxic acrolein. [14]Acrolein subsequently induces carbonyl stress to enhance DNA damage and inhibit GPx4 and DNA repair protein expression.However, the catalytic action of PAO is determined by oxygen concentration, which largely limits their antitumor effect in oxygen-poor tumor tissues.On the other hand, PTT alone can only play a limited therapeutic role due to the multiple defense mechanisms of tumors.Remarkably, APH+NIR treatment controlled the growth of tumors to the utmost extent.The slowest growth trend of tumors was observed in the APH+NIR group.The primary and distant tumor volume was inhibited to <200 mm 3 and <300 mm 3 respectively after the tumor inoculation for 21 days (Figure 4F,G).This verified the most significant antitumor effects of APH+NIR treatment, indicating the excellent synergistic effect of the combination of carbonyl stress and AIE-mediated PTT.Acrolein staining of tumor tissue on day 21 after treatment further confirmed our hypothesis (Figure 4I).Survival rates of the mice were also monitored.The mice treated with PBS+NIR and APH groups all died within 45 days (Figure 4H), while PAO and AH+NIR treatment prolonged the lifespan of tumor-bearing mice, representing 50-55 days of the median survival time.
As expected, mice in APH+NIR treatment displayed the highest survival rate within 60 days, revealing an obvious therapeutic outcome.

In vivo antitumor immune responses
To further evaluate DC maturation from ICD-induced tumors, lymph nodes and spleens were collected and homogenized lymphoblastic suspensions were co-stained with CD80 and CD86 antibodies as markers of DC maturation.The proportion of mature DC (CD80 + CD86 + ) was then measured by flow cytometry.Consistent with previous in vitro results, treatment with PAO and AH+NIR significantly increased the proportion of mature DC.The DC maturity rate of the APH+NIR treatment group was more than 1.5 times higher than that of AH+NIR, indicating that APH combined with carbonyl stress and PTT could effectively induce more mature DC (Figure 5A,B). [41]Recent studies have demonstrated that ICD effects promote intratumoral invasion of T lymphocytes. [3,42,43]It is proposed that mature DC induced by APH+NIR treatment could present antigen to T lymphocytes and stimulate immune response.Therefore, tumor-infiltrating CD8 + T cells were then detected by flow analysis.The proportion of CD8 + T cells in distant tumors in the APH+NIR group reached 24.03%, much higher than that in the PBS group (10.03%; Figure 5C,E).In addition, a significant increase in CD8 + T cell (represented as red fluorescence) infiltration was also observed from immunofluorescence assay in tumor tissues of mice treated with APH+NIR, indicating that APH+NIR treatment maximally activated CD8 + T lymphocytes (Figure 5D).These results confirmed that APH+NIR treatment effectively enhanced the invasion and activation of CD4 + and CD8 + T cells in tumors.In addition, as shown in Figure S7, the presence of B220 + CD8 + T cells indicated that B cells functioned as antigen-presenting cells to process and present antigens to T cells in the APH+NIR group, which elicited the potent antitumor immune response. [44,45]It is particularly noteworthy that as direct evidence of B cells presenting antigens to T cells, the proportion of B220 + CD8 + T cells indicated the interaction between T cells and B cells in the tumor.MDSCs are a heterogeneous population of immature cells of bone marrow origin that are recruited into tumor regions during tumorigenesis, infiltrating, and expansion, ultimately suppressing innate and adaptive immune responses through multiple T cell dysfunction. [46,47]4T1 breast cancer is a transplantable tumor cell line with high tumorigenicity and invasiveness, making it an animal model suitable for researchers to conduct breast cancer experiments.In fact, 4T1 is one of the best-studied mouse tumors with regard to immunology and anti-tumor immune response.Ostrand-Rosenberg et al. showed the critical role of MDSCs in this particular tumor. [48]Their study also showed that even without inflammatory stimulation, the proportion of MDSC in 4T1 tumor tissue reached 30%, indicating the existence of an immunosuppressive microenvironment in 4T1 tumor tissue.In addition, many studies have shown that reducing the MDSC ratio in 4T1 tumor tissue can effectively improve the effectiveness of tumor immunotherapy. [49,50]herefore, MDSCs can be targeted to regulate the immune response of APH (Figure 5F).Compared with other treatment groups, APH+NIR treatment significantly reduced the intratumoral frequency of MDSCs (CD11b + Gr1 + ).Furthermore, serum samples from mice were collected and the levels of tumor necrosis factor-α (TNF-α) and immune-related cytokine interferon-γ (IFN-γ) were measured by enzymelinked immunosorbent assay.Benefiting from ICD induced by APH+NIR, the secretion of TNF-α and IFN-γ was promoted, indicating that it effectively simulated the proliferation of cytotoxic T lymphocytes and enhanced the effect of systemic anti-tumor immunotherapy (Figure 5G,H).To sum up, APH-mediated combination therapy successfully evoked systemic immunity by promoting T-cell infiltration.
Immunomemory is another important criterion for providing long-term protection against tumor recurrence and rechallenge. [51]To study the anti-tumor rechallenge and recurrence effect of APH, another mice model was established accordingly.The primary tumor in the right hind leg of mice was resected after the abovementioned treatment groups and the mice were rechallenged with secondary 4T1 tumors in the left hind leg (Figure 6A).The volume of recurrence and rechallenge 4T1 tumor were recorded at certain time intervals.As shown in Figure 6C,E,G, the PBS+NIR group showed rapid tumor recurrence in mice, and the APH group slightly slowed tumor growth, while PAO alone and AH+NIR significantly inhibited tumor growth.Surprisingly, no tumor recurrence was detected in mice treated with APH+NIR.It was then observed that secondary homotype tumors invaded and grew in mice that received different treatments after surgically removing the primary tumors.(Figure 6D,F,G).4T1 tumors in the control group inoculated again grew rapidly, while the rechallenge tumors were significantly suppressed in mice pretreated with APH+NIR, indicating its efficacy in preventing tumor rechallenge.In addition, the APH+NIR group significantly extended the lifespan of mice with a high survival rate (80%) in 60 days (Figure 6B).This suggested that APH+NIR treatment elicited a durable immune response against rechallenge and recurrent tumors.The induction of systemic immune response was further evaluated by measuring the typical cytokines IFN-γ and TNF-α in serum (Figures S8 and S9).As expected, APH+NIR treatment continuously promotes IFN-γ and TNF-α secretion in serum compared to the control groups.For example, the blood concentration of TNF-α in the APH+NIR group was 2.5 times higher than that in the PBS group, indicating that APH+NIR successfully promotes pro-inflammatory and antitumor activity.Memory T cells are known to be essential for providing rapid protection against re-infection by pathogens and tumor recurrence.To assess the effects of long-term anti-tumor immunity, we studied the frequency of TCM (CD3 + CD8 + CD62L + CD44 + ) cells in collected blood samples of different treated mice (Figure 6H,I).The frequency of TCM increased significantly in the APH+NIR group and the proportion of TCM in the APH+NIR treated mice was almost 2.8 times higher than that of the control group.We can conclude that functional memory T cells produced by the APH+NIR group may be very beneficial for long-term protection from tumor invasion.In addition, we found that Ki-67 staining of the tumor slices with the APH combination treatment showed the most severe cell damage post, while the other five control treatment groups only exhibited partial cell damage on corresponding tumor slices (Figure 6J).We further constructed a 4T1 lung metastasis model and used the APH system for treatment.It was found that the number of lung metastases was reduced (Figure S10).Collectively, these results demonstrated that APH+NIR can effectively enhance anti-tumor immune response and induce long-term immune memory effect, thus preventing tumor recurrence, rechallenge, and metastasis.Meanwhile, the biosafety of the treatment group was further validated.Hematoxylin and eosin staining sections of major organs (heart, liver, spleen, lung, and kidney) showed no significant pathological morphological changes.Blood biochemical analysis showed high blood compatibility and no difference between groups, collectively indicating that the prepared APH had minimal systemic toxicity during treatment (Figures S11 and S12).All of these excellent results suggested that our fabricated APH with combinational therapeutic effect would be a potential and safe approach to not only suppress tumor growth but also show exciting results of inhibiting rechallenge and recurrence.

CONCLUSIONS
In summary, a thermal responsive APH hydrogel was smartly designed to exert PTT and induce carbonyl stress simultaneously for evoking antitumor immunity.Demonstrated by in vitro and in vivo experiments, the APH system effectively relieves tumor hypoxia via mild PTT, enhances PAO-mediated carbonyl stress therapy, and finally promotes cancer immunotherapy.The synergistic therapeutic effect was verified through a significant inhibitory effect on the distant tumor, enhanced immune memory, and effective suppression of postoperative recurrence and rechallenge.Considering the limitation of intratumoral injection for potential clinical application of tumor treatment, the promising therapeutic effect of the APH system prompts us to design an advanced nano hydrogel system, such as dendritic nano gel or N-Isopropylacrylamide, to load AIE phototheranostic agents and combinational drugs for intravenous administration.This APH system paves a strategy for developing clinical application scenarios, addressing urgent issues of the immunogenic environment in cancer patients.

A C K N O W L E D G M E N T S
We are grateful for the financial support from the National Natural Science Foundation of China (82002779), the Guangxi Natural Science Foundation under Grant No. 2023GXNSFBA026137, and the China Postdoctoral Science Foundation (2022M710853).Thanks to the AIE Institute (www.aietech.org.cn) for providing some AIE materials and technical assistance.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

E T H I C S S TAT E M E N T
The animal experiments were carried out according to the protocol approved by the Ministry of Health in the People's Republic of PR China and were approved by the Administrative Committee on Animal Research of the Shenzhen People's Hospital.

S C H E M E 1
Schematic illustration of near-infrared aggregation-induced emission nanoparticles boosting tumor carbonyl stress for enhanced immunotherapy of breast cancer.

F
I G U R E 1 (A) Chemical structure of 2TT-oC26B.(B) Photoluminescence (PL) spectra of 2TT-oC26B in dimethyl sulfoxide (DMSO)/water mixtures with varied volume fractions of water (f w ).(C) α AIE curves of 2TT-oC26B in DMSO/water mixtures with different f w .(D) Transmission electron microscopy (TEM) images of aggregation-induced emission (AIE) nanoparticles (NPs).Inset is the dynamic light scattering (DLS) distribution of AIE NPs in an aqueous medium.(E) Absorption and emission spectra of aggregation-induced emission (AIE) NPs in aqueous medium.(F) Photothermal stability of AIE NPs and indocyanine green (ICG) (50 mM) in aqueous solution during four on/off irradiation cycles with an 808 nm laser.(G) Scanning electron microscopy (SEM) image of the APH.(H) Temperature-dependent Rheological curves for the APH.(I) Temperature changes of APH at various AIE NPs concentrations under a 5 min irradiation from 808 nm laser at 0.5 W/cm 2 .(J) The IR image of the prepared APH and during laser irradiation.(K) The plasma amine oxidase (PAO) release profile in APH with or without 808 nm laser irradiation.Black arrows indicate irradiation time points.L: 808 nm laser.The absorption of BCA at 562 nm was recorded.(L) The generation of H 2 O 2 by APH (Containing 20 μg/mL PAO and different concentrations of spermine) was measured by ultraviolet-visible (UV-vis) absorbance of chromogenic tetramethylbenzidine (TMB) in the presence of horseradish peroxidase (HRP).The absorption of TMB at 652 nm was recorded.Data are shown as the mean ± SD, n = 3. F I G U R E 2 (A) Fluorescence images and (B) intensity of acrolein in 4T1 cancer cells treated with different formulations.Scale bars: 10 μm.The concentration of plasma amine oxidase (PAO) is 20 μg/mL.The cells were stained with an anti-acrolein antibody and anti-mouse IgG (H&L): FITC secondary antibody.λ ex : 488 nm, λ em : 525 nm.(C) Fluorescence images and (D) Flow cytometry analysis of reactive oxygen species (ROS) in 4T1 cancer cells treated with different formulations.Scale bars: 10 μm.The concentration of PAO is 20 μg/mL.λex : 488 nm, λ em : 525 nm.(E) Viability of 4T1 cells treated with different formulations in the presence of 350 μM spermine.The concentration of PAO is 20 μg/mL.(F) Viability of 4T1 cells treated with APH+NIR at PAO concentration in the presence of 350 μM spermine.(G) Flow cytometry analysis of cell apoptosis in 4T1 cells incubated with different formulations.Data are shown as the mean ± SD, n = 3; Student's t-test.(***p < 0.001).ofAPH+NIR on 4T1 cells increased with the increase of APH concentration.All these results indicated that APH had an excellent tumor cell-killing effect under laser irradiation, which could be attributed to the synergistic effect of mild PTT and carbonyl stress induced by the production of acrolein and H 2 O 2 .To investigate the mechanism of tumor cell death induced by APH+NIR, Annexin-V/PI assay was performed by flow cytometry.As shown in Figure2G, PAO and AH+NIR groups showed a higher percentage of cell apoptosis compared with PBS and APH groups.4T1 cells treated with APH+NIR had the highest apoptosis level, indicating that the tumor cell death induced by APH-mediated combination therapy under laser irradiation was mainly related to apoptosis.
, 4T1 cells were inoculated into the right side as the primary tumor and the left side as the abdominal wall tumor to establish a bilateral tumor model.When the tumor on the right side reached about 200 mm 3 , the mice were divided randomly into 5 different groups (each group included 5 mice): (1) PBS+NIR (808 nm, 0.5 W/cm 2 , 5 min); (2) APH; (3) PAO; (4) AH+NIR; (5) APH+NIR.The concentration of PAO is 2 mg/kg.The treatment outcome of each group was assessed by monitoring the mice's body weight and tumor volume every 3 days.The primary and distant tumors grew rapidly in the PBS+NIR group and expanded to 1027.4 and 799.8 mm 3 , respectively, on day 21 posttreatment (Figure 4D,E and Figure

F I G U R E 4
(A) Schematic illustration of the studies of 4T1 tumor therapy.(B) Pemonidazole (PIMO) stained tumor tissues after indicated treatments.λ ex : 488 nm, λ em : 525 nm.(C) Quantification of tumor hypoxia for different groups shown in (B).(D) Evolution of the primary and (E) distant tumor volumebearing mice after various treatments.(F) Digital photos of primary and (G) distant tumors in different groups after treatment.(H) Survival curves after treatment.(I) Acrolein staining in tumor tissues after 21 days of treatment scale bars are 40 μm.λ ex : 488 nm, λ em : 525 nm.Data are shown as the mean ± SD, n = 5; ***p < 0.001; Student's t-test.F I G U R E 5 (A) Flow cytometry analysis of treatment-induced dendritic cell maturation in the lymph nodes and (B) spleen.(C) CD4 + CD8 + T lymphocytes in distant tumors.(D) CD8 staining analyses of distant tumor tissues treated with various formulations.Scale bars: 40 μm.λ ex : 495 nm, λ em : 519 nm.(E) Quantitative analysis of CD8 + T lymphocytes in distant tumors and mature dendritic cells (DC) cells in lymph nodes.(F) Flow cytometry analysis of intratumoral myeloid-derived suppressor cells (MDSCs) (CD11b + Gr1 + ) at days 7. (G) Enzyme-linked immunosorbent assay (ELISA) analysis of the levels of proinflammatory cytokines IFN-γ and (H) Tumor necrosis factor alpha (TNF-α) in serum of mice isolated at the end of treatments.Data are shown as the mean ± SD, n = 3. **p < 0.01, ***p < 0.001; Student's t-test.F I G U R E 6 (A) Schematic illustration of the studies of 4T1 tumor rechallenge and recurrence.(B) Survival curves after treatment.(C) Recurrence and (D) rechallenge 4T1 tumor growth.(E) Digital photos of recurrence and (F) rechallenge tumors in different groups after treatment.(G) Tumor weight of rechallenge and recurrence tumors at the end of treatments.(H) Representative flow dot plots and (I) the data of TCM in blood on day 14 after the different treatments measured by flow cytometry.(J) Ki-67 staining analyses of rechallenge and recurrence tumor tissues treated with various treatments.Scale bars: 40 μm.λ ex : 590 nm, λ em : 617 nm.Data are shown as the mean ± SD, n = 5; ***p < 0.001; Student's t-test.