GLP was extracted from the fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst by Hangzhou Johncan International, the content was about 30%. ICG was purchased from Dalian Meilun Biotech (Dalian, China). Bombyx mori Cocoons were purchased from Bozhou Naxi Pharmaceutical (Bozhou, China). Lithium Bromide was purchased from Sigma-Aldrich (St Louis, MO, United States). Dialysis bag (3,500 Da) and HE staining kit were purchased from Solarbio (Beijing, China).

SF was prepared as the method previously described (Kim et al., 2017). Bombyx mori Cocoons were cut into pieces and soaked in 0.02 M Na₂CO₃ solution. Then boiled for 1 h to remove sericin while stirring constantly. Washed 3 times and dried at 65°C. After drying, cocoons were dissolved in a water bath with 9.3 M LiBr solution at 60°C for 3 h. Then, filtrated twice with filter paper. Finally, dialyzed with dialysis bag (MWCO 3500) against 2,000 ml ultra-pure water for 72 h to remove the LiBr. The SF solution was further purified by freeze-drying to obtain silk fibroin freeze-dried powder, sealed and stored in a drying box.

ICG-SF-Gel was prepared by ultrasonic oscillation. Briefly, ICG solution (2 mg/ml) and SF solution (60 mg/ml) were mixed in an equal proportion. Then, ultrasound (100 W) was performed until turbidity appeared. Finally, the ultrasound was stopped and standing for 30 min.

GLP was prepared by heating and dissolving. Briefly, 1 g of GLP powder was added to 6 ml of pure water, heated and dissolved in a constant temperature water bath at 60°C, centrifuged at 2,000 rpm for 5 min, then the precipitation was discarded to get about 6 ml of GLP solution.

To investigate the photothermal effect of ICG and ICG-SF-Gel in vitro, 200 μl of ICG solution (1 mg/ml) and ICG-SF-Gel were added into a 48-well cell culture plate and irradiated with 808 nm NIR laser (1.0 W/cm²) for 300 s. During the irradiation, the temperature was recorded. SF (30 mg/ml) and Ultra-pure water were used as control. To investigate the photothermal circle stability, the temperature changes of four groups were measured by light on/off (5 min/30 min) three times.

Mouse hepatoma 22 (H22) cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology (Zqxzbio, ZQ0109, China). The cells were cultured in RMPI Medium 1,640 (Gibco, C11875500BT, United States), supplemented with 10% fetal bovine serum (Gibco, 1600044, United States), 1% penicillin, and streptomycin (Solarbio, P1400, China) at 37°C under a circumstance containing 5% CO₂. Adjust H22 cell strains to 1 × 10⁷ cells/ml, then injected 0.2 ml into the abdomen of male BALB/C mice for 6–8 days. The ascites cells were passaged three times for usage. The cell concentration was 1 × 10⁷ cells/ml.

All the data were presented as mean ± SD. All statistical analysis was carried out using GraphPad Prism 7.04 for Windows. For all the immunohistochemical staining analysis, the Student’s t-test was performed to determine the significance of differences between two groups, and one-way analysis of variance (ANOVA) was employed for multiple group comparison. Survival curves were plotted using the Kaplan-Meier method and compared between groups using the Log-rank (Mantel-Cox) test. Two-way ANOVA was used for time-temperature curves. The temperature curve, relative tumor growth curve and body weight curve were analyzed by two-way ANOVA. In all tests, p < 0.05 was considered statistically significant. Statistical significance was expressed by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

SF was prepared as the method previously described. The concentration of SF solution was approximately 3 wt%. ICG solution (2 mg/ml) and SF solution (60 mg/ml) were mixed in an equal proportion. Then, ultrasound (100 W) was performed until turbidity appeared. Finally, the ultrasound was stopped and standing for 30 min to obtain ICG-SF-Gel.

The UV spectrum of ICG (10 μg/ml) and ICG-SF solutions was investigated and results were displayed in Figure 2A. The Ultra-pure water and SF solution exhibited no absorption peaks at 400–900 nm. ICG and ICG-SF solutions exhibited a strong characteristic absorption peak at 779 nm, indicating that the successful encapsulation of ICG in the SF solution and SF did not affect the UV absorbance of ICG.

In our previous study, we carried out the characterization experiments of ICG-SF-Gel, which proved the safety of ICG-SF-Gel (Xu et al., 2018; Yao et al., 2020).

GLP was extracted from the fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst by Hangzhou Johncan International, the content was about 30%. It was prepared by heating and dissolving. Briefly, 1 g of GLP powder was added to 6 ml of pure water, heated and dissolved in a constant temperature water bath at 60°C, centrifuged at 2000 rpm for 5 min, then the precipitation was discarded to get about 6 ml of GLP solution.

The photothermal effect of ICG-SF-Gel, ICG (1 mg/ml) in vitro was evaluated for 300 s and results were shown in Figures 2B–I.

To explore a more suitable concentration of ICG, we carried out experiments on the photothermal effect of ICG at different concentrations and 1 mg/ml was selected. (Figure 2B). The result showed that the photothermal effect of the continuous 808 nm NIR radiation on Ultra-pure water and SF (30 mg/ml) was negligible. By contrast, after radiation for 10 s, the temperature of ICG (1 mg/ml) began to rise rapidly, after 260 s the temperature was elevated up to 65°C. ICG-SF-Gel also showed a temperature rising after radiation. The temperature of ICG-SF-Gel began to rise rapidly after 10 s, then rose and remained at 69°C after 160 s. Compared with the ICG solution, ICG-SF-Gel has a higher temperature and faster heating rate (Figure 2B).

To investigate the photothermal circle stability in vitro, the temperature change of ICG (1 mg/ml) and ICG-SF-Gel under the discontinuous 808 nm NIR irradiation mode (ON = 5 min, OFF = 30 min, three cycles, 1 W/cm²) was performed (Figures 2C–I) and the similar results were obtained (Figure 2G). The photothermal effect of the first radiation on the temperature of Ultra-pure water and SF (30 mg/ml) was negligible. By contrast, after radiation for 20 s, the temperature of ICG (1 mg/ml) began to rise rapidly, after 200 s the temperature was elevating up to 60°C. ICG-SF-Gel also exhibited a temperature rising after the first radiation, the temperature began to rise rapidly after 20 s, and after 140 s the temperature was elevated up to 60°C. Compared with the ICG solution, a higher temperature and faster heating rate were observed for ICG-SF-Gel (Figure 2G). As expected, the photothermal effect of discontinuous 808 nm NIR irradiation mode on the temperature of Ultra-pure water and SF (30 mg/ml) were negligible (Figures 2C,D). Compared with the ICG solution, the heating rate and maximum platform temperature of ICG-SF-Gel decreased more obviously (Figures 2E,F). In Figure 2E, the temperature rise of the three radiations was the same in the first 40 s, indicating that the photothermal stability of ICG is good. Besides, the first radiation also consumed ICG, but less than the ICG-SF-Gel group, which was still enough to support the ICG consumption of the second radiation, so the second photothermal curve did not decrease. However, in the third radiation, the content of ICG has been consumed too much, resulting in a decrease in the temperature in the third photothermal curve. In Figure 2F, SF-Gel could control the release of ICG and aggregate ICG, resulting in more ICG consumption, more temperature rise, and faster heating rate during the first radiation. Then, also due to the controlled release and aggregation effect of SF-Gel on ICG, the consumption of ICG was higher. Therefore, in the second and third laser irradiations, the content of ICG was lower, the heating rate and the maximum platform temperature of the ICG-SF-Gel group decreased more obviously. Therefore, in the follow-up animal experiments, we had an injection of ICG before every radiation to ensure the stability of the effect of PTT.

Overall, these results suggested that ICG could convert light to heat, and ICG-SF-Gel may converge and accelerate the photothermal effect so that ICG can achieve a faster and stronger photothermal effect.

To confirm the photothermal transformation of ICG/ICG-SF-Gel in vivo, and to stimulate the immune response of the body, we treated the mice with 808 nm NIR laser (1 W/cm²) every 24 h intervals for 3 days. The maximum temperatures of the tumor area and thermo-graphic images were recorded for 90 s (Figure 3A and Supplementary Material).

As shown in Figures 3C–F, on the first day, the tumor area temperature of H22 group mice increased slowly from 36.6 to 40.2°C. By contrast, the tumor area temperature of H22 + ICG group mice rapidly increased from 35.6 to 55.5°C after irradiation for 10 s, then kept increasing at a relatively fast rate. Finally, the temperature was elevated up to 75.5°C. In the ICG-SF-Gel group, the tumor area temperature rapidly increased from 35.6 to 56.8°C after 10 s, then kept increasing at a relatively fast rate. Finally, the temperature was elevated up to 81.4°C. Compared with the H22 + ICG group, the tumor area temperature of the ICG-SF-Gel group increased faster and higher, which may be due to the aggregation effect of SF-Gel on ICG, so that ICG can achieve a faster and stronger photothermal effect.

In the 3 days NIR irradiation experiment, the tumor area temperature of H22 group mice increased slowly from 36 to 40°C (Figure 3C). The heating rate of H22 + ICG group mice was faster and higher on day 2 than day 1, then decreased on day 3, but still faster and higher than day 1 (Figure 3D). The heating rate of ICG-SF-Gel group mice was faster and higher on day 2 than day 1, then decreased on day 3, but still slightly faster and higher than day 1 (Figure 3E). However, in the experiment of discontinuous NIR radiation in vitro, the heating rate of ICG and ICG-SF-Gel groups decreased slightly, and the maximum platform temperature kept decreasing. The reason for this difference may be the effect of skin color and tissue on NIR heat absorption. As shown in Figure 3B, after irradiation, the tumor areas of the mice were burned and the tissue moisture content decreased, making it easier to heat up. Besides, the pigmentation in the tumor area increases the absorption of light energy, thus making the thermal effect of the laser more significant.

Compared with the H22 + ICG group, the tumor area temperature of the ICG-SF-Gel group increased faster and higher on day 1, but slightly slower and lower on day 2 and day 3 (Figures 3F–H). It is consistent with results of discontinuous NIR radiation in vitro, which may be due to the aggregation effect of SF-Gel on ICG. Due to the higher consumption of ICG in the ICG-SF-Gel group mice on day 1, the photothermal effect of ICG-SF-Gel group mice was slightly weaker than that of H22 + ICG group mice on day 2 and day 3.

All in all, the results of in vivo experiments also demonstrated that ICG could convert light to heat, and ICG-SF-Gel may converge and accelerate the photothermal effect of ICG so that ICG can achieve a faster and stronger photothermal effect.

The Schematic illustration of ICG/ICG-SF-Gel-based PTT and GLP immunotherapy to inhibit distal tumor growth is shown in Figure 4A. After 7 days of ultra-pure water or GLP intragastric administration, 0.2 ml of the H22 cells (1 × 10⁷ cells/ml) were subcutaneously injected into the back of the left hindlimb of each mouse as the primary tumors. When the primary tumors reached 60 mm³, H22 + ICG + Laser and H22 + ICG + GLP + Laser groups were intratumorally injected with 0.1 ml ICG (1 mg/ml), H22 + ICG-SF-Gel + Laser and H22 + ICG-SF-Gel + GLP + Laser groups were intratumorally injected with 0.1 ml ICG-SF-Gel. After injections, the tumor site of mice in five groups was irradiated for 90 s. The maximum temperatures of the tumor area and thermo-graphic images were recorded for 90 s. The irradiation and temperature thermal imaging were performed again each day for the next 2 days. In the 3 days experiment, the tumor area temperature of mice injected with ICG or ICG-SF-Gel under laser irradiation quickly rose to 55°C in 30 s, which was high enough to effectively ablate tumors. While in the H22 group, the tumor area temperatures of mice exhibited no significant increase during irradiation (Figure 4B).

To investigate the antitumor efficiency of ICG/ICG-SF-Gel-based PTT and GLP immunotherapy in hepatoma-bearing mice, the primary tumor size was measured daily. The relative primary tumor volumes were calculated as V/V₀ (V₀ is the tumor volume when ICG/ICG-SF-Gel were injected). As illustrated in Figure 4C, ICG/ICG-SF-Gel-based PTT and GLP immunotherapy significantly inhibited the growth of the primary tumors. Compared with the H22 group, the increase of relative primary tumor volume in H22 + ICG + Laser and H22 + ICG-SF-Gel + Laser groups were slowed down significantly, while that in H22 + ICG + GLP + Laser and H22 + ICG-SF-Gel + GLP + Laser groups slowed down more obviously, suggesting that ICG/ICG-SF-Gel-based PTT inhibited the growth of primary tumors and GLP could enhance the inhibitory effect.

Besides, GLP mainly plays the role of an immunomodulator to enhance the inhibition of tumors. GLP alone can’t directly inhibit tumor growth, and GLP needs to be dissolved by heating during intragastric administration. In this process, GLP may be decomposed into glucose-based monosaccharides, which has been proved in the literature published in science that glucose can promote the growth of the primary tumor (Goncalves et al., 2019), which may lead to the relative primary tumor volume in the GLP + H22 group is higher than that in the H22 group alone. Laser alone can’t kill the H22 tumor, but due to the mild temperature stimulation caused by the laser, it accelerates the blood circulation around the tumor and promotes the growth of the primary tumor, which may lead to the relative primary tumor volume in the GLP + Laser group is higher than that in the H22 group alone (Figure 4C).

Eight days after the primary tumor inoculation, 0.2 ml of the H22 cells (1 × 10⁷ cells/ml) were subcutaneously injected into the back of the right forelimb of each mouse as the distal tumors with no direct treatments. To further investigate the distal antitumor efficiency of ICG/ICG-SF-Gel-based PTT and GLP immunotherapy in hepatoma-bearing mice, the distal tumor size was measured daily. The relative distal tumor volumes were calculated as V/V₀ (V₀ is the tumor volume of the third day after distal tumor inoculation). As shown in Figure 4D, ICG/ICG-SF-Gel-based PTT and GLP immunotherapy significantly inhibited the growth of distal tumors. Compared with the H22 group, the increase of relative tumor volume in H22 + ICG + Laser and H22 + ICG-SF-Gel + Laser groups were slowed down significantly, while that in H22 + ICG + GLP + Laser and H22 + ICG-SF-Gel + GLP + Laser groups slowed down more obviously, suggesting that ICG/ICG-SF-Gel-based PTT inhibited the growth of distal tumors and GLP could enhance the inhibitory efficacy.

However, unlike primary tumors, there was no significant difference in distal tumors between the H22 + GLP and H22 + laser groups and the H22 group. This may be due to the different mechanisms of combined treatment of primary and distal tumors. The inhibitory effect of combination therapy on the primary tumor is mainly the direct killing effect on tumor cells, while the inhibitory effect on the distal tumor is achieved through the distal effect brought by immune function. In the H22 + GLP group, because the effect of combination therapy on the distal tumor is achieved through immune function, the promoting effect of sugar on tumor growth may be offset by strong immune function. In the H22 + Laser group, the laser did not directly stimulate the distal tumor, so it did not affect the growth of the distal tumor.

To evaluate the health status of mice, the body weight was measured daily. The relative body weight was calibrated by normalizing the initial body weight (at day 0) to 1. The relative body weight was plotted at regular intervals and considered a surrogate for evaluation of systemic well-being. There were no significant body weight changes in the seven groups during the experiment (Figure 4E), indicating the fewer side effect of the treatments.

To evaluate the survival of mice, the mortality of mice was recorded daily. As displayed in Figure 4F, ICG-based PTT and GLP immunotherapy significantly resulted in improving the survival time. Compared with the H22 group, the H22 + ICG + Laser group exhibited a significant increase in improving the survival time, and in the H22 + ICG + GLP + Laser group, the survival time increased more obviously, indicating that ICG-based PTT prolonging the survival of mice bearing H22 tumors, and GLP can further prolong the survival time. Meanwhile, Consistent with ICG-based PTT and GLP immunotherapy, ICG-SF-Gel-based PTT and GLP immunotherapy significantly improved the survival time. Compared with the H22 group, the H22 + ICG-SF-Gel + Laser group exhibited no significant increase in improving the survival time, but in the H22 + ICG-SF-Gel + GLP + Laser group, the survival time increased significantly, indicating that ICG-SF-Gel-based PTT prolonging the survival of mice with the combination of GLP.

As a promising natural source of immunomodulatory, GLP exerts the antitumor action by stimulating immune function (Sohretoglu and Huang, 2018; Wang et al., 2018). We introduced GLP immunotherapy to improve the distal antitumor efficiency of tumor-associated antigens produced in situ after photothermal ablation of primary tumors, and to inhibit the development of tumors.

To explore the antitumor mechanism of ICG/ICG-SF-Gel-based PTT and GLP immunotherapy, the tumors from different treatment groups of mice 7 days after the distal tumor inoculation were collected for HE staining and immunohistochemical staining.

First, to explore the effect of photothermal ablation, the morphological and pathological changes of primary tumors from different treatment groups of mice 7 days after the distal tumor inoculation were analyzed through HE staining. As shown in Figure 5A, the H22 group displayed robust tumor tissues with no necrosis and had rich normal capillaries. Binucleated cells with large nuclei and dense chromatin were frequently observed, indicating rapid tumor growth. In contrast, cell shrinkage and nuclear fragmentation were evident in H22 + ICG + Laser, H22 + ICG-SF-Gel + Laser, H22 + ICG + GLP + Laser and H22 + ICG-SF-Gel + GLP + Laser groups, revealing that PTT based on ICG/ICG-SF-Gel could induce tumor necrosis. Besides, more tumor necrosis and fragmentation in nuclei with evident cytoplasmic separation from nuclei were found in H22 + ICG + GLP + Laser and H22 + ICG-SF-Gel + GLP + Laser groups, indicating that GLP could enhance the photothermal ablation efficacy. These results suggested that PTT based on ICG/ICG-SF-Gel could achieve the effect of photothermal ablation and GLP could enhance the photothermal ablation efficacy.

Then, the morphological changes, pathological changes, and cell proliferation of distal tumors after different treatments were analyzed through HE staining and Ki-67 immunohistochemical staining. As shown in Figure 5B, the H22 group had large areas of confluent tumor cells with little or no tumor tissue necrosis. Consistent with the primary tumors, cell shrinkage and nuclear fragmentation were observed in distal tumors after PTT based on ICG/ICG-SF-Gel, and GLP-assisted PTT led to more tumor necrosis. Besides, compared with ICG-based PTT, PTT based on ICG-SF-Gel led to more tumor necrosis, indicating that PTT based on ICG-SF-Gel have a stronger inhibitory effect on distal tumor growth than ICG-based PTT. Ki-67 is a nuclear protein expressed in proliferating mammalian cells, suggesting the high proliferation potential of the tumor cells (Sobecki et al., 2016). Ki-67 immunohistochemical staining results revealed that PTT based on ICG/ICG-SF-Gel inhibited the cell proliferation of distal tumors and GLP enhanced the inhibition effect. Compared with ICG-based PTT, ICG-SF-Gel-based PTT enhanced the inhibition of cell proliferation (Figures 5C,G). All these results suggested that PTT based on ICG/ICG-SF-Gel could inhibit the cell proliferation of distal tumors and GLP could enhance the inhibition efficacy.

Some studies have revealed that GLP reduces the expression of some signaling molecules in the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways which play a key regulatory function in proliferation at both gene and protein levels (Suarez-Arroyo et al., 2013). In the follow-up work, we will explore whether this signaling pathway is involved in our study.

Caspase-3 is a key executioner in apoptosis which is involved in the growth stimulation (Huang et al., 2011). BAX is a pro-apoptotic gene, it has been proposed that GLP enhances the anti-cancer effects by up-regulation of BAX (Huang et al., 2010). Bcl-2 is a proto-oncogene, which can inhibit apoptosis, mainly because bcl-2 regulates a variety of cell apoptosis-related protein activity, such as by Caspase-3 (Ola et al., 2011). It was reported that GLP induced HUVECs apoptosis directly by decreasing anti-apoptotic protein Bcl-2 expression (Cao and Lin, 2006). It is recognized that GLP can induce apoptosis in cancer cells by regulating the expression of bcl-2 (Martínez-Montemayor et al., 2011). To explore whether cell apoptosis plays a role in ICG/ICG-SF-Gel-based PTT and GLP immunotherapy, the distal tumors from different treatment groups of mice 7 days after the inoculation of the distal tumor were analyzed through immunohistochemical staining of Caspase-3, BAX, and bcl-2. Compared with the H22 group, distal tumors treated with PTT based on ICG/ICG-SF-Gel displayed a more obvious expression of Caspase-3 and BAX, and GLP increased the expression, demonstrated that GLP could increase cell apoptosis of distal tumors induced by PTT based on ICG/ICG-SF-Gel. Compared with ICG-based PTT, ICG-SF-Gel-based PTT increased the expression of Caspase-3 and BAX, indicating that ICG-SF-Gel-based PTT could induce cell apoptosis of distal tumor more effectively than ICG-based PTT (Figures 5D,E,H,I). Compared with the H22 group, distal tumors treated with PTT based on ICG/ICG-SF-Gel displayed lower expression of bcl-2 and GLP decreased the expression, demonstrated that PTT based on ICG/ICG-SF-Gel could inhibit the expression of apoptosis inhibitor gene and GLP could enhance the inhibitory effect. Compared with ICG-based PTT, ICG-SF-Gel-based PTT decreased the expression of bcl-2, indicating that ICG-SF-Gel-based PTT could inhibit the expression of apoptosis inhibitor genes more effectively than ICG-based PTT (Figures 5F,J). GLP may up-regulate the expression of BAX, inhibit the expression of Bcl-2, and then activate the expression of Caspase-3, thus enhancing the apoptosis of distal tumors. All in all, these results pointed out that ICG-SF-Gel-based PTT could increase cell apoptosis of distal tumors more effectively than ICG-based PTT, and GLP could enhance the pro-apoptosis efficacy.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a transmembrane glycoprotein member expressed on endothelial cells, which has been shown to play an important role in angiogenesis (Cao et al., 2009). Fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) were also involved in angiogenesis, and FGF-2 was reported to stimulate endothelial cell growth and enhances angiogenesis (Bikfalvi, 1999). Besides, GLP has been reported to inhibit angiogenesis by reducing the expression of VEGF (Sohretoglu and Huang, 2018). Therefore, angiogenesis of distal tumors from different treatment groups of mice 7 days after the inoculation of the distal tumor was analyzed through immunohistochemical staining of PECAM-1, VEGFA, and FGF-2. Compared with the H22 group, distal tumors treated with PTT based on ICG/ICG-SF-Gel displayed lower expression of PECAM-1 and VEGFA, and GLP decreased the expression more obviously, suggested that PTT based on ICG/ICG-SF-Gel could inhibit angiogenesis and GLP could enhance the inhibitory efficacy (Figures 6A,B,D,E). Compared with ICG-based PTT, ICG-SF-Gel-based PTT decreased the expression of PECAM-1, VEGFA, and FGF-2, indicating that ICG-SF-Gel-based PTT could inhibit angiogenesis more effectively than ICG-based PTT. (Figures 6A–F). These results demonstrated that ICG-SF-Gel-based PTT could inhibit angiogenesis more effectively than ICG-based PTT, and GLP could enhance the anti-angiogenic efficacy. GLP may inhibit angiogenesis by inhibiting the proliferation of vascular endothelial cells and the production of PECAM-1, VEGFA and FGF-2, thus inhibiting the growth of distal tumors.

It has been established that the immune system plays an important role in tumorigenesis (Baxevanis et al., 2009), and photothermal immunotherapy is a promising treatment combining PTT and immunotherapy (Yata et al., 2017). PTT utilizes heat generated by optical absorbing agents under NIR light to dissolve tumor cells, induced apoptosis or necrosis of tumor cells based on hyperthermia (Zhang et al., 2014). Then, tumor cell necrosis releases tumor-associated antigens, triggering specific antitumor immunity and clearing the residual tumor (Guo et al., 2014). As a promising natural source of immunomodulatory, GLP activates the antitumor immune response and enhances immune function (Sohretoglu and Huang, 2018; Wang et al., 2018). TNF-α is an important marker in the activation of cellular immunity (Xiang et al., 2015), CD68 is a pan-marker of macrophages, IL-1β and IL-6 are important proinflammatory cytokines secreted by M1 macrophages (Zong et al., 2019). IL-13 is a Th2 cytokine that plays a critical role in a novel immunoregulatory pathway in which natural killer T cells suppress tumor immunosurveillance (Terabe et al., 2000; Terabe et al., 2004). It was reported that GLP could activate bone marrow-derived macrophages to produce immunomodulatory substances, such as TNF-α, IL-1β and IL-6 (Wang et al., 1997; Zhang et al., 2010).

To investigate whether the immune system plays an important role in ICG/ICG-SF-Gel-based PTT and GLP immunotherapy, the distal tumors from different treatment groups of mice 7 days after the distal tumor inoculation were analyzed through immunohistochemical staining of TNF-α, CD68, IL-1β, IL-6 and IL-13. Compared with the H22 group, distal tumors treated with PTT based on ICG/ICG-SF-Gel displayed a more obvious expression of TNF-α and GLP increased expression, suggesting that PTT based on ICG/ICG-SF-Gel could increase cellular immunity and GLP could enhance cellular immunity induced by PTT based on ICG/ICG-SF-Gel. Compared with ICG-based PTT, ICG-SF-Gel-based PTT increased the expression of TNF-α, indicating that ICG-SF-Gel-based PTT could induce cellular immunity more effectively than ICG-based PTT (Figures 7A,F). Compared with the H22 group, distal tumors treated with PTT based on ICG-SF-Gel displayed a more obvious expression of CD68 and GLP increased the expression of CD68 induced by ICG/ICG-SF-Gel based PTT, indicating that PTT based on ICG-SF-Gel could increase the infiltration of macrophages and GLP could enhance the infiltration of macrophages induced by ICG/ICG-SF-Gel based PTT (Figures 7E,J). Compared with the H22 group, distal tumors treated with PTT based on ICG/ICG-SF-Gel displayed a more obvious expression of IL-1β and GLP increased the expression of IL-1β induced by ICG-based PTT, indicating that PTT based on ICG/ICG-SF-Gel could increase the infiltration of M1 type macrophages and GLP could enhance the infiltration of M1 type macrophages induced by ICG-based PTT (Figures 7B,G). Compared with the H22 group, distal tumors treated with PTT based on ICG/ICG-SF-Gel displayed a more obvious expression of IL-6 and GLP increased expression, suggesting that PTT based on ICG/ICG-SF-Gel could increase the infiltration of M1 type macrophages and GLP could enhance the infiltration of M1 type macrophages induced by PTT based on ICG/ICG-SF-Gel (Figures 7C,H). Compared with the H22 group, distal tumors treated with PTT based on ICG/ICG-SF-Gel decreased the expression of IL-13 and GLP decreased the expression more obviously, indicating that PTT based on ICG/ICG-SF-Gel could inhibit the down-regulation of tumor immunosurveillance induced by IL-13 and GLP could enhance the inhibitory efficacy (Figures 7D,I). All these results suggested that the immune system plays an important role in ICG/ICG-SF-Gel-based PTT and GLP immunotherapy and GLP could enhance immune function. GLP may promote the secretion of IL-6, IL-1β and TNF-α by activating CD68 macrophages, reduce the inhibition of IL-13 secreted by natural killer T cells on tumor immune surveillance, strengthen the immune function of the body, and thus inhibit the growth of distal tumors.

Due to COVID-19 and other reasons, our experimental animals and cells suffered severe losses, which limited our research methods to immunohistochemical staining, unable to use other research methods to support the experimental results. Therefore, In the follow-up work, we will use more experimental methods to support the experimental results and conduct further research.