Dipalmitoyl phosphatidylcholine (DPPC), cholesterol (CHO), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000 (DSPE-PEG-2000) were obtained from A.V.T. Pharmaceutical Co., Ltd. (Shanghai, China). Chlorin e6 (Ce6) and Perfluorohexane (PFH) were purchased from J&K Science Ltd. (Beijing, China). 1,3-Diphenylisobenzofuran (DPBF) was obtained from PYTHONBIO (Kaifeng, China), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Sigma-Aldrich. Anti-Calreticulin antibody was supplied by Abcam (Cat. #ab92516), Calcein AM was obtained from Beyotime (Cat. #C2012), PI was obtained from Solarbio (Beijing, China), CCK-8 was purchased from Dojindo Molecular Technologies, Inc. (Japan). The aPD-1 antibody was purchased from BioXcell (Cat. #BE0146). Antibodies for flow cytometry, including anti-CD45-PerCP (Cat. #103130), anti-CD3-FITC (Cat. #100204), anti-CD8-APC (Cat. #100711), anti-CD4-APC (Cat. #100412), anti-FoxP3-PE (Cat. #320008), anti-CD11c-FITC (Cat. #117306), anti-CD80-PE (Cat. #104708), anti-CD86-APC (Cat. #105012), anti-CD8-PE/Cy7 (Cat.# 100721), anti-CD4-BV650 (Cat. #100469), anti-CD44-PE (Cat. #103008), and anti-CD62L-APC (Cat. #104412) were obtained from Biolegend. Anti-CD8 antibody was supplied by Cell Signaling Technology (Cat. #98941S). Ki67 antibody was obtained from Servicebio Technology Co. Ltd., (Wuhan, China). ELISA kits were purchased from Dakewe Biotech Co., Ltd (Shenzhen, China).

The PCL@O₂ was synthesized by filming-rehydration method. Briefly, A 6:3:1 weight ratio of DPPC:CHO: DSPE-PEG (total of 10 mg) and 1.0 mg Ce6 were dissolved in 2 mL CH₂Cl₂ in a 25 mL flask. Subsequently, the prepared film was hydrated and 100 μL of PFH was added into above suspension under sonication via a sonicator. Finally, PCL@O₂ could be harvested by bubbling O₂ gas in above PCL suspension. The resulted nanodroplets was stored at 4 °C. Ultimately, the Ce6 liposome@O₂ nanodroplets (CL@O₂) was prepared using the similar process except the addition of PFH.

The Hydrodynamic diameter and Zeta potential of synthesized PCL@O₂ were evaluated by via Nano ZS90 (Nano ZS, Malvern, USA). The morphology was obtained via transmission electron microscopy (TEM- JEM-2100F). These loading effect of Ce6 was evaluated by standard curve method via UV-vis spectrometer (Synergy HTX, BioTEK Co., USA).

Moreover, ROS generation of PCL@O₂ triggered by ultrasonic irradiation was determined by DPBF, whose UV-via absorbance peak can be quenched by generated ROS. Briefly, DPBF (3 mM, 160 μL) and different formulation (PCL@O₂, CL@O₂ and H₂O) were added into pre-deoxygenated water and the mixed solution (2 mL, Ce6 2.5 μg/mL, DPBF 240 μM) was subsequently irradiated by US (1.0 MHz, 1.6 W/cm², 50% duty cycle) for 15 min. Then, the absorbance peak of DPBF was measured and recorded by UV–vis spectrophotometer.

The O₂ loading and ultrasound-responsive O₂ release of oxygenated PCL@O₂ were investigated by monitoring dissolved O₂. Briefly, 1 mL different formulation (PCL@O₂, CL@O₂ and H₂O) was added into 7 ml pre-deoxygenated water. After 2 min, the ultrasound irradiation (1.0 MHz, 1.6 W/cm², 50% cycle) was carried out for 4 min. The concentration of dissolved O₂ was detected in a sealed chamber equipped with portable dissolved oxygen meter (JPB-607A, INESA & Scientific Instrument Co., Ltd, Shanghai, China) in real time.

The murine colon adenocarcinoma cancer cell line MC38 was obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (China). The MC38 cell line was cultured in dulbecco’s modified eagle medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin under 5% CO₂ at 37 °C.

The intracellular ROS generation was evaluated by DCFH-DA. Briefly, after adhering in 24 wells plates, the MC38 cells were incubated with PCL@O₂ or CL@O₂ nanoparticles at Ce6 concentration of 5 μg/mL for 4 h. Subsequently, the cells were exposed to ultrasound irradiation (1.0 MHz, 1.6 W/cm², 50% duty cycle) for 1 min, incubated with DCFH-DA (10 μM) for 30 min and then observed by fluorescence microscope (ECLIPSE TS2R FL, Nikon, Japan). Quantitative analysis of the fluorescence intensity was performed using Image J software.

The cytotoxicity of SDT was evaluated by CCK-8 assay. After the completion of incubation and irradiation described previously, the cells were further incubated for another 2 h. The cell viability was assessed using CCK-8, and the OD 450 nm value was measured by a multimode reader (Synergy HTX, BioTEK Co., USA). Additionally, the cell viability was evaluated by live/dead cell staining. Similarly, the treated MC38 cells were stained with Calcein-AM and PI for 30 min, and observed by fluorescence microscope (ECLIPSE TS2R FL, Nikon, Japan). CRT expression on the treated cell surface was also observed to assess immunogenic cell death caused by SDT. Briefly, after fixation with 4% paraformaldehyde, the treated cells were blocked with 5% BSA, incubated with the anti-calreticulin antibody overnight and then with secondary antibody for 1 h. After further counterstaining with DAPI, the MC38 cells were visualized by fluorescence microscope. Semi-quantitative analysis of the fluorescence intensity was performed using Image J software.

Bone marrow cells were isolated from 6-week-old C57BL/6 mice and cultured according to the established protocol to induce immature bone-marrow-derived dendritic cells (BMDCs). The induced immature BMDCs were plated in the lower compartment of a transwell system (1×10⁶ cells per well). The MC38 cells treated with various treatments including incubation with nanodroplets for 4 h, irradiation to ultrasound (1.0 MHz, 1.6 W/cm², 50% duty cycle, 1min) were put into the upper compartment of the transwell system. After co-incubation for 24 h, the treated BMDCs were stained with anti-CD11c-FITC, anti-CD80-PE and anti-CD86-APC and then collected for flow cytometric assessment.

Female C57BL/6 mice (6-8 weeks old, 18-20 g) were provided by Guangdong Medical Laboratory Animal Centre. All animal experiments and operations were approved by Institutional Animal Care and Use Committee of the Sun Yat-sen University (approval number: SYSU-IACUC-2022-001655).

To develop bilateral subcutaneous MC38 tumor bearing mice, 40 μL and 120 μL of MC38 cell suspension (1×10⁷/mL PBS) were subcutaneously injected into the left and right flank, respectively. Treatments were initiated after 2 weeks, when the long diameter of the right tumor reached about 0.6 cm. The C57BL/6 mice bearing bilateral subcutaneous MC38 tumors were randomly divided into five groups receiving various treatments: (1) Control, (2) RFA, (3) RFA + aPD-1, (4) RFA + SDT, (5) RFA + SDT + aPD-1. The radiofrequency ablation treatments were performed using a radiofrequency electrode with an active tip of 1 cm. The tip was inserted percutaneously into the center of the right tumor for about 1 min depending on the actual tumor size, until the temperature reached 60°C within the tumor. For the SDT therapy, the PCL@O₂ ([Ce6] = 667 μg/mL, 100 μg/injection) was intravenously injected three times on day 2, 5 and 8 after RFA administration. Subsequently, the primary tumor (the ablated one) of treated mice were irradiated with ultrasound (1.0 MHz, 1.6W/cm², 30%, 8 min) at 12 and 24 h after PCL@O₂ injection. For PD-1 blockade, 200 μg anti-PD-1 was administered through intraperitoneal injection every three days for a total of three times.

In vivo fluorescence imaging was performed to evaluate the accumulation of nanodroplets at tumor site and major organs. When the MC38 tumor volume grew up to 100 mm³, the in vivo fluorescence image of tumor-bearing mouse was acquired as control (0 h). After that, PIL@O₂ (1 mg/kg IR-780) was intravenously injected via tail vein to reveal the biodistribution of nanodroplets. The fluorescence images were acquired at different time points (2, 4, 6, 9, 12, and 24 h) by Lumina XR Series III system (Perkin Elmer, USA, IR780: Ex, 720 nm; Em, 790 nm). 24 hours after the administration of PIL@O₂, the major organs (heart, liver, spleen, lung and kidney) and tumor nodule were collected for ex vivo fluorescence imaging. Subsequently, the fluorescence signal intensities were quantified and calculated with living imaging system (IVIS lumina).

Tumor diameters were measured using calipers every 2 days till 30 days after RFA treatment. The tumor volume was calculated using the formula, V = length × width²/2. For immune responses investigation, primary and distant tumors were harvested on the 10^th day and divided into three parts. A portion of the tumor tissues were minced and digested with 125 μg/mL collagenase and 12.5 μg/mL DNAse at 37 °C for 15 min, then ground and filtered with 1% FBS PBS buffer to prepare tumor cell suspension for flow cytometry analysis. A portion of the tumors were fixed in 4% paraformaldehyde for histology and the remaining tumors were prepared for tissue homogenates in PBS to analyze cytokines by Enzyme-linked immunosorbent assay (ELISA).

The above-processed cell suspension was stained with fluorochrome-conjugated antibodies for the evaluation of CD8⁺ T cells (CD45⁺CD3⁺CD8⁺), CD4⁺ T cells (CD45⁺CD3⁺CD4⁺) and Tregs (CD45⁺CD3⁺CD4⁺FoxP3⁺) for 30min and then analyzed using flow cytometry. Tumor-draining lymph nodes were also collected and made into single-cell suspension to analyze dendritic cells (DCs, CD11c⁺CD80⁺CD86⁺) and central memory T cells (Tcm, CD44⁺CD62L⁺) in CD8⁺ T cells or CD4⁺ T cells.

Fixed tumor tissues in 4% paraformaldehyde were then embedded in paraffin, sectioned, dewaxed and rehydrated, and retrieved the antigen. Then, tumor tissue sections were conducted hematoxylin-eosin (H&E), and IHC staining by Ki67 and CD8. Eventually, staining images were obtained using microscope.

To further investigate the level of related cytokines (TNF-α and IFN-γ), the tumor tissue homogenates were assessed using the ELISA kits according to the instructions.

Major organs (heart, liver, spleen, lung, and kidney) from each group were obtained and fixed in 4% paraformaldehyde for H&E staining. Blood samples were collected, and the serological analyses containing hepatic and renal functional markers were carried out following the manufacturer’s protocol.

Prism 8.0 (GraphPad Software) was used to analyze data. Statistical analyses were conducted using two-tailed unpaired t test. Survival rates were analyzed by the Kaplan-Merier method and compared with log-rank test. Data were expressed as mean ± standard deviation (SD)/standard error of mean (SEM). P < 0.05 was considered to be statistically significant.

The SDT nanodroplet, PCL@O₂, with a PFH core and a lipid membrane were synthesized by typical reverse evaporation method ( Figure 1 ). Phospholipid as one of the most excellent biocompatibility materials was used as shell for loading hydrophobic agents Ce6 (sonosensitizer). Additionally, the O₂-loaded nanodroplet, PCL@O₂, was obtained by bubbling O₂ gas. As light scattering (DLS) illustrated, PCL@O₂ displayed an averaged hydrodynamic diameter of 230.17 ± 4.31 nm (PDI = 0.15 ± 0.02) ( Figure 2A ), and an averaged zeta potential of 0.47 ± 0.05 mV ( Supplementary Figure 1 ). As monitored by the transmission electron microscopic (TEM) image ( Figure 2B ), the PCL@O₂ showed a spherical morphology and homogeneous size. Additionally, the loading content of Ce6 was calculated to be 6.53 ± 0.05%.As shown in Figure 2C , the PCL@O₂ nanodroplet was well dispersible in aqueous solution, and no aggregation in PBS containing 10% fetal calf serum (FBS) within 48 h.

To investigate the ROS production capacity of PCL@O₂ under US irradiation, 1,3-diphenyliso- benzofuran (DPBF) were employed as molecular probe to monitor the ROS generation (13, 17). As shown in Figures 2D, E , the absorbance peak of DPBF in PCL@O₂ solution decreased starkly under US irradiation, while the absorption peak of DPBF in CL@O₂ decreased slowly. After US irradiation for 15 min, the decreased DPBF concentration of PCL@O₂ group and CL@O₂ nanodroplet was 55.3 ± 3.8% and 22.8 ± 5.8%, respectively. In comparison, the change of DPBF peak absorption in control group was negligible.

The O₂-loaded PCL@O₂ was synthesized as an augmented SDT nanoplatform. As reported, O₂ can be loaded and stabilized by PFH for about two weeks, which significantly prolong the in vivo application time (18). As shown in Figure 2F , the dissolved oxygen concentration of PCL@O₂ was obviously increased to 10.8 mg/L. In comparison, only a slight increase (up to 5.8 mg/L) of dissolved oxygen concentration was observed in CL@O₂ group. After ultrasound irradiation, the PCL@O₂-containing solution showed a dramatic increase of oxygen concentration to about 13.3 mg/L, which was higher than that of the CL@O₂ group. Thus, results documented the good O₂ loading ability and US-induced O₂ release of PCL@O₂, suggesting that PCL was potentially for delivering O₂ to tumor in vivo and releasing it site-specifically under focal US irradiation.

The ROS generation inside MC38 cancer cells of Ce6-loaded nanodrug with US irradiation was measured with DCFH-DA, a ROS indicator. The Ce6 induced ROS with US irradiation can oxidate DCFH to strong fluorescent product DCF (oxidation product of DCFH), therefore showing the ROS level. As observed under confocal laser scanning microscopy, MC38 tumor cells incubated with PBS, CL@O₂ and PCL@O₂ without US irradiation or PBS with US irradiation showed negligible DCF fluorescence ( Figure 3A , Supplementary Figure 2A ), indicating US irradiation and sonosensitizer are both necessary for ROS generation. Compared with CL@O₂, US irradiation induced higher green fluorescence intensity in cells incubated with PCL@O₂, showing the robust synergy of O₂-filled PFH in ROS generation. The live and dead cell viability assays were then carried out to evaluate the cytotoxicity effect of the generated ROS. Cells incubated with different nanodrugs without US irradiation or PBS with US irradiation showed strong green fluorescence, exhibiting good cell activity. In comparison with CL@O₂ + US treatment, cells incubated with PCL@O₂ + US showed much stronger red fluorescence and weaker green fluorescence, stating that PCL@O₂ + US could result in more effective cytotoxicity ( Figure 3B , Supplementary Figure 2B ). ROS-induced cytotoxicity of nanodrug was further evaluated with CCK-8 analysis. Without US irradiation, cells incubated with PCL@O₂(5 μg/mL) showed a good viability above 97%, indicating the nanodrug possessed a good biocompatibility. After irradiated with ultrasound, PCL@O₂ + US at same concentration induced more apoptosis and a much lower viability (39%) than that of CL@O₂ + US (49%) ( Figure 3C ).

Owing to SDT can generate intracellular ROS to induce ICD, immunofluorescence staining of CRT in MC38 cells, a common ICD marker that could stimulate antigen presentation and induce immune response (19, 20) was conducted. As shown in Figure 3D and Supplementary Figure 2C , cells treated with nanodrugs alone or US irradiation alone exhibited negligible green fluorescence. In comparison, the expression levels of CRT of PCL@O₂ + US group was much stronger than that of CL@O₂ + US. These results clearly proved that PCL@O₂ induced enhanced SDT could significantly amplify ICD to potentially provoke an immune response.

To initiate tumor immunity, antigen presentation to cytotoxic T lymphocytes by antigen presenting cells (APCs) is indispensable, especially DCs. Activated DCs can capture tumor antigens and travel to lymphoid tissues where they activate tumor antigen-specific T lymphocytes. Maturation of DCs is a prerequisite for inducing effective anti-cancer immune responses. The ability of DCs to activate T cells correlates with their ability to mount anti-tumor immune responses (21, 22). Therefore, promoting the activation and maturation of DCs after RFA is quite critical.

In this study, transwell system was used to evaluate the maturation of DCs by culturing immature BMDCs with MC38 cells receiving various treatments. Because DCs start to mature upon antigen engagement, the differential effects on mature DCs may explain the effect of differential treatments on DCs in vivo. As shown in Figure 3E , oxygen self-enriching SDT by PCL@O₂ + US clearly increased the number of CD11c⁺CD80⁺CD86⁺ mature BMDCs compared to other groups. The above results illustrated that the robust effect on DCs maturation may be due to that SDT promoted cancer cell death through apoptosis and necrosis, and DCs engulfed stressed and necrotic tumor cells.

The pharmacokinetics of nanodrug in MC38 tumor-bearing mice was evaluated in vivo by intravenous injection of PIL@O₂ (1 mg/kg IR780). IR780 absorbs in the NIR region at 720 nm and can be captured with Lumina XR Series III system. The fluorescent intensities increased gradually post injection of nanodrug ( Figures 4A, B ). At the time point of 24 h, the tumor and major organs were collected, and fluorescence signal from IR780 was collected for quantification analysis. As shown in Figures 4C and D , the accumulation of nanodrug was obviously higher in the tumor than the other organs. This increased tumor accumulation may be promoted by enhanced permeability and retention (EPR) effect.

In further in vivo experiment, C57BL/6 mice were inoculated with MC38 on bilateral flanks, respectively, to evaluate the synergistic anticancer effect of SDT and anti-PD-1therapy in vivo. When the long diameter of the right tumor reached about 0.6 cm, the mice were randomly divided into the following five groups: control, RFA, RFA + aPD-1, RFA + SDT, and RFA + SDT + aPD-1. As shown in Figure 5A , insufficient RFA treatment was performed in the right tumor, which was defined as primary tumor, while the left tumor was defined as distant tumor. The volume of both primary and distant tumors in the RFA group of mice treated with iRFA was not significantly different from that of the control group, probably due to the smaller extent of ablation and the continued rapid growth of the residual tumors after ablation. All treatments with nanodrugs showed obvious therapeutic effects in both primary and distant tumors, and tumor growth was most effectively inhibited in mice treated with SDT and aPD-1 after iRFA. On day 10 after iRFA treatment, tumor volume of primary tumors in the RFA+ SDT+ aPD-1 group was 270.88 ± 82.06 mm³, and distant tumor reached 205.32 ± 66.13 mm³. In comparison, primary tumors grew to 1183.97 ± 209.82 mm³, 1073.08 ± 212.74 mm³, 706.02 ± 165.61 mm³ and 625.05 ± 150.44 mm³ in the control group, RFA group, RFA + aPD-1 group and RFA+SDT group, respectively ( Figures 5B, D ). A similar trend was observed for distant tumors, with tumor volume of 744.07 ± 170.24 mm³, 652.48 ± 125.56 mm³, 509.59 ± 140.65 mm³ and 425.53 ± 76.78 mm³ in the control group, RFA group, RFA + aPD-1 group and RFA + SDT group, respectively ( Figures 5C, E ). Consistent with the results of tumor growth inhibition, the RFA + SDT + aPD-1 treatment most effectively prolonged the survival rate of mice ( Figure 5F ). These results indicated that the combination of augmented SDT and aPD-1 raise a highly effective systemic anti-tumor immune response against both primary and distant tumor even with iRFA burdens, compared with the slightly inhibition of aPD-1 therapy alone.

To further validate the synergistic therapeutic efficacy, H&E, Ki-67 and TUNEL staining assays were performed on day 10 after iRFA in the five groups. As shown in Supplementary Figure 3 , compact tumor cells with an intact structure were showed in the control group, and necrosis with destructive cells was observed in the groups treated with iRFA, while there were still surviving tumor cells around the ablation zone in RFA group. The results of TUNEL staining were consistent with the results of H&E staining. These results were further confirmed by the proliferation biomarker Ki-67 in iRFA zone of tumors ( Figure 5H ). Ki-67 was considerably reduced in each treatment group compared with control group, and significantly abridged by combined therapy of SDT and aPD-1. Therefore, combination therapy is superior to treatment alone in limiting cell proliferation in MC38 tumors after iRFA.

Additionally, the biosafety of PCL@O₂ nanodrug was evaluated through body weight changes, H&E staining of the major organs, and blood biochemical markers. No significant changes in the body weights of the mice in different groups were observed after treatments ( Figure 5G ), and no obvious tissue damage or inflammation in heart, liver, spleen, lung, and kidney appeared in mice receiving different treatments ( Supplementary Figure 4 ). In addition, the levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin (TBIL) indicating the liver functions, as well as creatinine (CREA) and UREA indicating the kidney functions, were not elevated and within the normal range in mice receiving different treatments ( Supplementary Figure 5 ), indicating that combination therapy did not injure the liver and kidney.

To further investigate the influence on immune microenvironment after different treatments from different angles, we collected tumor-draining lymph nodes, primary and distant tumors on the 10th day after first treatment for immunofluorescence staining, flow cytometry and cytokine analyses. Immunofluorescence staining of CRT in primary tumors was firstly conducted. As shown in Supplementary Figure 6 , CRT staining on the cell membrane was clearly observed in combination treatment group, indicating PCL@O₂ with enhanced SDT plus aPD-1 therapy could amplify ICD by CRT expression to potentially provoke an immune response after iRFA.

Chronic exposure of damage-associated molecular patterns (DAMPs) by ICD could attract receptors and ligands on DCs and activate immature DCs to transition to a mature phenotype (19). Therefore, the maturation of DCs (CD11c⁺CD80⁺CD86⁺) in tumor draining lymph nodes was analyzed by flow cytometry after different treatment. As shown in Figure 6A , owing to the enhancement of SDT and aPD-1, the percentage of DCs maturation in RFA + SDT + aPD-1 group was 9.2%, which was significantly higher than that of control group (6.6%). Flow cytometry gating scheme for immune cells are described in Supplementary Figure 7 .

Then, the percentage of tumor-infiltrating mature T cells (CD45⁺CD3⁺) in various treatment groups were measured, because tumor infiltration of T cells plays a key role in anti-tumor immunity. As shown in Figures 6B and D , the percentage of T cells in RFA + SDT + aPD-1 group obviously increased to 14.4 ± 1.9% and 9.6 ± 1.6% in primary and distant tumors respectively, which was nearly 2.2-fold and 1.6-fold higher than control group respectively. The percentage of T cells in combination therapy group was also significantly higher than RFA, RFA + aPD-1 and RFA + SDT group. A similar trend was seen in the percentage of CD8⁺ cytotoxic T lymphocytes (CTLs). As shown in Figures 6C and F , the SDT plus aPD-1 treatment after iRFA resulted in a much higher percentages of CTLs in T cells (32.3 ± 2.7%) than treatments using RFA alone (20.2 ± 1.4%), RFA + aPD-1 (22.9 ± 1.0%), RFA + SDT (21.1 ± 2.6%), and PBS (15.4 ± 0.6%) in primary tumors. Consistent results were obtained in distant tumor tissues. The percentage of CTLs in combination treatment group (29.2 ± 1.6%) was significantly higher than other groups ( Figures 6E, G ). Tumor infiltration of the CD8⁺ T cells was further confirmed by immunofluorescent staining of tumor tissue sections. Comparison with the rare tumor infiltration of CD8⁺ T cells in the control group, CD8⁺ T cells was obviously increased via combination therapy in both primary and distant tumors ( Figures 6H, I ). In particular, the RFA + SDT + aPD-1 treatment showed the best effect to promote the tumor infiltration of CD8⁺ T cells. This may be due to increased mature DCs facilitating antigen presentation to induce more cytotoxic T lymphocytes (CTLs). These results showed that the oxygen self-enriching nanodrug may mediate effective Ce6-based SDT synergistic with aPD-1 treatment to enhance the tumor infiltration of CD8⁺ T cells.

Considering that TNF-α and IFN-γ produced by immune cells can induce the secretion of pro-inflammatory cytokines for a strong anti-tumor immunity (23, 24), the levels of TNF-α and IFN-γ in primary and distant tumors were also detected on the 10th day after first treatment by ELISA to verify the activation of the tumor-cell killing of immune cells. As shown in Figures 6J–M , compared with the control treatment or RFA alone, combination therapy of SDT and aPD-1 after iRFA induced a significant increase of both TNF-α and IFN-γ levels in bilateral tumors. Moreover, the level of IFN-γ induced by SDT plus aPD-1 therapy after iRFA was even much higher than that of the RFA + aPD-1 or RFA + SDT, which implied a strong synergistic effect of SDT and aPD-1 to activate T cells and initiate effective anti-tumor immune response after iRFA.

The immune response to tumor antigen presence within the tumor, especially in the liver cancer, where the RFA therapy mostly applied, could lead to the systemic suppression of antitumor immunity (10). This immune suppression was associated with the coordinated activation of Tregs and modulation of intratumoral CD11b⁺ monocytes. Thus, the attraction of Tregs was characterized by the positive expression of both CD4 and Foxp3 using flow cytometry. As shown in Figures 7A, B and E , although there was an increase in total CD4 T cells in primary tumors of RFA + SDT + aPD-1 group, the proportion of Tregs in CD4 T cells significantly deceased in combination therapy group, compared to control, RFA alone, RFA + aPD-1 or RFA + SDT group. A similar trend also appeared in the distant tumor ( Figures 7C, D , Supplementary Figure 8 ). It is worth noting that there was an increase of Tregs in primary tumor of RFA group, which indicating that insufficient RFA therapy could induce reduction in systemic antitumor immunity and restrain immunotherapy efficacy via Tregs-mediated immune suppression. Interestingly, this increase of Tregs after iRFA can be diminished by optimized SDT therapy and further eliminated by combination of aPD-1 therapy.

Exhaustion of effector T cells following tumor antigen clearance, which results in their short persistence, can limits the efficacy of immunotherapy after iRFA. Since the long-lived Tcm persist longer and produce stronger effector functions that have the potential to control tumor recurrence after RFA, their generation is an important goal for the combination treatment (25). Thus, the proportion of CD8⁺ Tcm (CD3⁺ CD8⁺ CD62L⁺ CD44⁺) and CD4⁺ Tcm (CD3⁺ CD4⁺ CD62L⁺ CD44⁺) in tumor-draining lymph nodes was measured to explore the long-term immune memory effect. As shown in Figures 7F and H , the RFA + SDT + aPD-1 combination therapy resulted in a significant increase in CD8⁺ Tcm, compared with the treatment of control, RFA alone, RFA + SDT or RFA + aPD-1. Interestingly, the proportion of CD4⁺ Tcm also obviously increased in the RFA + SDT + aPD-1 group ( Figure 7G ), which may also produce more antigen-specific CD4⁺ T cells. These antigen-specific CD4⁺ T cells are reported to enhance the efficacy of anti-tumor CD8⁺ T cell responses, and mediate long-lived protection against subsequent tumor recurrence (26). These results indicated that the synergistic SDT and aPD-1 therapy after iRFA can elicit effective and long lasting anti-tumor response.