The Synergistic Anti-tumor Effect of Iodine-125 Low-dose-rate Brachytherapy and Anti-PD-1 Therapy on Lung Cancer in Mice

Background: Radiotherapy (RT) when combined with anti-PD-1 therapy has a signicant effect, but RT fractionation and dose impact the effects of this combined therapy. Iodine-125 particle implantation ( 125 I RPI) is a hyperfractionated low-dose-rate brachytherapy. Its impact on the tumor immune microenvironment and the ecacy of 125 I RPI combined with anti-PD-1 therapy are unknown. In this study, we evaluated the effectiveness of 125 I RPI combined with anti-PD-1 therapy and their impact on tumor immunity. Methods: A Lewis lung cancer (LLC) mouse model was established and radioactive iodine-125 particles were implanted into the tumors. Tumor tissues were obtained six and 12 days after particle implantation, and the expression of PD-1/PD-L1 was detected by ow cytometry. On day 0, LLC cells were injected subcutaneously into the right hindlimb (primary tumor) and left ank (secondary tumor) of mice. On day 10, the mice were randomly divided into PBS, α-PD-1, 125 I RPI, and α-PD-1+ 125 I RPI groups. On day 22, tumor tissues were extracted from the mice. The proportion of immune cell subsets in the tumor immune microenvironment was detected by ow cytometry, and the primary and secondary tumor volumes were monitored. Results: After 125 I RPI, the expression of PD-L1 on tumor cells was upregulated (P < 0.0001), and the proportion of CD8+ PD-1+ T cells also increased (P = 0.0001). 125 I RPI combined with anti-PD-1 therapy synergistically inhibited primary (P = 0.0139) and secondary (P = 0.0494) tumor growth. The ow cytometry results showed that the combination therapy could increase the proportion of CD8+ T cells (P = 0.0055) and decrease the proportion of T regulatory cells (P < 0.0227) in the tumor microenvironment. Survival analysis shows that the sequence of 125 I RPI and α-PD-1 affects ecacy, and early initiation of 125 I RPI is more benecial. Conclusion: combined with anti-PD-1 therapy can signicantly inhibit tumor growth and activate anti-tumor immunity, which present a promising approach for the of on day 12. The expression of on tumor cells also increased on day 12 after 125 I RPI. This indicates that with increasing time after implantation and radiation dose accumulation, T cell function is gradually impaired, limiting the immune activation effect of 125 I RPI. Our results show that 125 I RPI combined with anti-PD-1 therapy can signicantly inhibit the growth of the primary tumor and can also induce the abscopal effect, which can inhibit the growth of secondary tumors, whereas 125 I RPI treatment alone cannot produce the abscopal effect. We showed that combination therapy can reduce immune suppression and activate systemic anti-tumor immune responses.


Introduction
Radiotherapy (RT) when combined with anti-PD-1 therapy has a signi cant effect, but RT fractionation and dose impact the effects of this combined therapy. Iodine-125 particle implantation ( 125 I RPI) is a hyperfractionated low-dose-rate brachytherapy. Its impact on the tumor immune microenvironment and the e cacy of 125 I RPI combined with anti-PD-1 therapy are unknown. In this study, we used a mouse model of LLC to evaluate the effect of 125 I RPI on the PD-1/PD-L1 axis, the e cacy of 125 I RPI combined with anti-PD-1 therapy, and the effect of combined therapy on the proliferation and function of Tlymphocytes. We also investigated the effect of the treatment sequence on their e cacy to provide preclinical evidence for hyperfractionated brachytherapy combined with anti-PD-1/PD-L1 therapy.

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The synergistic anti-tumor effect of iodine-125 low-dose-rate brachytherapy and anti-PD-1 therapy on lung cancer in mice PD-1/PD-L1 is a major immunosuppressive molecule in the (1). PD-L1 expressed on tumor cells can bind PD-1 on activated T cells and leads to T cell exhaustion, thus preventing immune killing of tumor cells (2). Anti-PD-1/PD-L1 treatment can eliminate immunosuppression and activate anti-tumor immunity. Clinical studies have shown that anti-PD-1/PD-L1 therapy has been applied to a variety of advanced tumors with signi cant e cacy (3). Radiotherapy (RT) is an indispensable method used in the treatment of cancer. In recent years, increasing attention has been paid to the impact of RT on tumor immunity. Studies have found that RT can also reduce the size of tumors outside of the radiation eld; this is known as the abscopal effect (4). The abscopal effect occurs when RT enhances the anti-tumor immune response. RT can promote the expression of tumor-associated antigens and neoantigens (5)(6)(7), making the irradiated tumor an "in situ vaccine" (8). RT can also induce damage-associated molecular patterns, including changes in molecules such as ATP, calreticulin, and the high mobility group of B1 proteins that can promote antigen uptake and presentation (9)(10)(11). However, the abscopal effect is rare in clinical practice. This is because RT can upregulate the expression of immunosuppressive molecules such as PD-L1 and T regulatory cells (Tregs) (12,13). This negative regulatory effect on immunity means that the immune activation effect of RT is not su cient to disrupt the immunosuppressive state of the tumor microenvironment. Studies have shown that RT and anti-PD-1/PD-L1 therapy have a synergistic effect, and combination therapy can reverse the immunosuppressive state (14,15).
RT fractionation (conventional fractionated, hypofractionated, and single-dose) and dose have different effects on tumor immunity, and are key factors affecting the e cacy of RT when combined with immunotherapy (16,17). Dewan et al. showed that fractionated RT could induce the abscopal effect when combined with anti-CTLA-4 treatment, whereas single-dose RT did not induce the abscopal effect(16). 125 I particle implantation ( 125 I RPI) is a type of hyperfractionated low-dose-rate brachytherapy, which is widely used to treat various solid tumors, including cancer of the lung, pancreas, liver, prostate, bladder, and rectum (18)(19)(20)(21). Currently, the impact of 125 I RPI on the tumor immune microenvironment is poorly understood, and no studies exist on hyperfractionated RT combined with immunotherapy. In addition, most studies used external beam RT (EBRT) combined with immunotherapy (22)(23)(24), and studies of brachytherapy combined with immunotherapy are rare. In this study, we investigated its e cacy.
In this study, we used a mouse model of LLC to evaluate the effect of 125 I RPI on the PD-1/PD-L1 axis, the e cacy of 125 I RPI combined with anti-PD-1 therapy, and the effect of combined therapy on the proliferation and function of T-lymphocytes. We also investigated the effect of the treatment sequence on their e cacy to provide preclinical evidence for hyperfractionated brachytherapy combined with anti-PD-1/PD-L1 therapy.

Materials And Methods
Cell lines LLC cells were purchased from the Cell Bank at Shanghai Institute of Cell Biology, Chinese Academy of Science. LLC cells were cultured in DMEM high glucose medium with 10% fetal bovine serum (both from GIBCO, Thermo Fisher Scienti c, Waltham, MA, USA) and incubated in a 5% CO 2 incubator at 37 °C. All cells were cultured for a limited passage before implantation.
Mice and tumor challenge C57BL/6 female mice, aged 6-8 weeks, were purchased from Experimental Animal Center of Chongqing Medical University. Cells in logarithmic growth phase were collected, and a 1 × 10 6 cell suspension was injected subcutaneously (SC) into the right hindlimb (primary tumor) of mice on day 0, and the left ank (secondary tumor) on day 3. On day 10, when the tumor volume was ~ 200 mm 3 , mice were randomly assigned to the phosphate-buffered saline (PBS) group, the 125 I RPI group, the α-PD-1 group, or the 125 I RPI + α-PD-1 group. Tumor volume was measured and recorded with vernier calipers every 2-3 days; a tumor volume of (length × width 2 )/2 and tumor diameter exceeding 20 mm was taken as the endpoint of the survival analysis.
Tumor therapy 125 I RPI therapy: The mice were anesthetized by intraperitoneal (IP) injection of 1% pentobarbital at a dose of 50 mg/kg. After securing the mice, an 18 g needle was inserted into the center of the tumor 0.5 cm away from the tumor edge, and the depth of the needle was measured and marked. The needle core was withdrawn, and a radioactive 125 I particle (0.8 mci, 29.6 MBq) was implanted into the center of the tumor. A cotton ball was used to stop the bleeding once the needle was withdrawn. The mice were reared in single cages, one mouse per cage. The mice were observed, and their food intake, level of activity, and general condition were monitored, and the skin area around the tumor was assessed for signs of redness, swelling, and ulceration.
Anti-PD-1 treatment: α-PD-1 monoclonal antibody (mAb) (clone RMP1-14; Bio X Cell, Lebanon, NH, USA) was administered by IP injection at a dose of 200 µg/mouse, once every other day for a total of ve times.

Flow cytometry
The mice were sacri ced and the tumor tissues were cut into sections of < 3 mm in length with ophthalmic scissors. We added 2 ml DMEM high glucose medium containing 1 mg/ml collagenase IV   Fig. 2A and 2B). In addition, combination therapy inhibited the growth of secondary tumors, whereas monotherapy did not (P = 0.0410, anti-PD-1 vs. 125 I RPI + α-PD-1 = 2503.2764 ± 473.71 mm 3 vs.
1624.67 ± 155.10 mm 3 on day 22; Fig. 2C). As shown in Fig. 2D, the median survival time of the PBS, 125 I RPI, α-PD-1, and 125 I RPI + α-PD-1 groups were 20, 22, 26, and 34 days, respectively. Compared with 125 I RPI and α-PD-1, combination therapy resulted in signi cantly longer survival (P = 0.0026 and P = 0.0019, respectively; Fig. 2D). These results suggest that anti-PD-1 therapy can improve the e cacy of 125 I RPI and combination therapy can induce the abscopal effect and improve the prognosis in mice.

Combination Therapy Increases Proliferation And Activation Of Cd8 + tils
To determine whether combination therapy could activate anti-tumor immunity, we analyzed T lymphocytes in the TME. Mice treated with 125 I RPI or anti-PD-1 had a slightly increased proportion of CD8 + TILs (P = 0.4103 and P = 0.6318, respectively), but those treated with combination therapy had signi cantly increased numbers of CD8 + TILs (P < 0.05 compared with all other groups; Fig. 3A and 3B).
There was no difference in the proportion of CD4 + TILs among the groups (P = 0.1539, 125 I RPI + α-PD-1 vs. PBS; Fig. 3A and 3B). To evaluate the activity of CD8 + TILs, we analyzed the ability of CD8 + TILs to produce IFN-γ and TNF-α (Fig. 3C). There were approximately ve-fold more IFN-γ + CD8 + TILs in the 125 I RPI + α-PD-1 group than in the PBS group ( Fig. 3D; P = 0.0013), and six-fold more TNF-α + CD8 + TILs than in the PBS group ( Fig. 3D; P = 0.0002). These results showed that combined therapy could activate antitumor immunity and increase the number and activity of T cells in the TME. We also showed that the immune activation effect of 125 I RPI is achieved through PD-1 blockade.
Combined therapy reduces the number of Tregs in the TME Tregs can promote tumor growth by inhibiting T cell activation (25). Studies have found that RT can upregulate the expression of Tregs in the TME(13), but anti-PD-1 treatment can reduce the expression of Tregs (26). However, the effect of 125 I RPI and 125 I RPI combined with anti-PD-1 treatment on Tregs is unknown. To this end, we analyzed the proportion of Tregs and showed that 125 I RPI did not affect the proportion of Tregs (P = 0.2442). The proportion of Tregs decreased after anti-PD-1 treatment (P = 0.0051), but the decrease was more signi cant after the combined treatment ( Fig. 4A and 4B; P = 0.0227).
We further analyzed the ratio of CD8 + T/Tregs, which was signi cantly higher in the 125 I RPI + α-PD-1 group than in the other groups (P < 0.05 compared with all other groups; Fig. 4B). These results suggest that combination therapy could reshape T cell immunity and make the tumor immune microenvironment into one of immune activation.
The schedule is a critical determinant affecting the e cacy of combination therapy The sequence of RT and anti-PD-1 treatment affects the e cacy of the combined therapy. Previous studies showed that concurrent therapy is better than sequential therapy (12,(27)(28)(29). To determine whether administration of 125 I RPI or α-PD-1 mAb at different times affects the e cacy of combination therapy, we examined three different combination schedules: 125 I RPI followed after three days by α-PD-1 mAb (schedule A); concurrent administration of α-PD-1 mAb and 125 I RPI (schedule B); and α-PD-1 mAb followed after three days by 125 I RPI (schedule C) (Fig. 5A). The treatment start time of the three schedules was on day 10 after injection of the tumor cells. The median survival time for schedules B and C were 34 and 33 days, respectively, while schedule A reduced the survival time compared to 125 I RPI only (P = 0.0128, 22 days vs. 26 days; Fig. 5B). In schedule A, α-PD-1 mAb did not control tumor growth, demonstrating that when 125 I RPI was used at a later stage, the tumor burden was already too high. This could explain why schedule A was less e cient. These results suggest that initiating 125 I RPI earlier to delay tumor progression represents a more effective combination schedule.

Discussion
In this study, we used a lung cancer model to demonstrate PD-1/PD-L1 inhibition of the anti-tumor immune response caused by 125 I RPI. On day 6 after 125 I RPI, PD-L1 expression was upregulated, but PD-1 expression was not. On day 12, PD-L1 was further upregulated; PD-1 expression was also upregulated.
After 125 I RPI combined with anti-PD-1 therapy, the immunosuppressive state was reversed, the in ltration of CD8 + T cells in the TME increased nearly two-fold, and the proportion of Tregs reduced three-fold, which signi cantly inhibited growth of the primary and second tumors. In addition, we showed that the sequence of 125 I RPI and anti-PD-1 therapy affects the e cacy of the combined therapy, and delaying 125 I RPI reduces the e cacy of combination therapy.
PD-1 is a surface marker of exhausted T cells. Our results showed that the proportion of CD8 + PD-1 + TILs did not increase on day six after 125 I RPI, but increased signi cantly on day 12. The expression of PD-L1 on tumor cells also increased on day 12 after 125 I RPI. This indicates that with increasing time after particle implantation and radiation dose accumulation, T cell function is gradually impaired, limiting the immune activation effect of 125 I RPI. Our results show that 125 I RPI combined with anti-PD-1 therapy can signi cantly inhibit the growth of the primary tumor and can also induce the abscopal effect, which can inhibit the growth of secondary tumors, whereas 125 I RPI treatment alone cannot produce the abscopal effect. We showed that combination therapy can reduce immune suppression and activate systemic anti-tumor immune responses.
The results achieved were further veri ed by the ow cytometry results. The proportion of CD8 + TILs in the TME increased signi cantly following the combined therapy, and the ability of CD8 + TILs to secrete IFN-γ and TNF-α was also signi cantly enhanced. This indicated an increase in in ltrated T cells in the TME and, therefore, enhanced activity and killing ability. Schau et al. showed that a single dose of 15 Gy increased the expression of Tregs in mouse melanoma, but two fractions of 7.5 Gy reduced the expression of Treg (30). In our study, the proportion of Tregs did not increase or decrease after 125 I brachytherapy characterized by hyperfractionation, but decreased in combination with anti-PD-1 therapy. It is worth noting that the ratio of CD4 + TILs did not increase after the combined treatment. One explanation is that Tregs accounted for ~ 21% of CD4 + T cells in the PBS group, compared with ~ 7% in the 125 I RPI + α-PD-1 group. The decrease in Tregs resulted in no change in the total number of CD4 + T cells, which masked the increase of effector CD4 + T cells. Another explanation is that CD4 + T cells are not the effector cells of 125 I RPI when combined with anti-PD-1 therapy, so there was no increase. Many preclinical studies have found that CD4 + T cells are dispensable for RT combined with anti-PD-1 therapy, and depletion of CD4 + T cells did not affect the e cacy of the combined treatment, whereas CD8 + T cells are essential (12,14).
Dovedi et al. found that administration of α-PD-L1 mAb seven days after completion of RT did not produce an effective anti-tumor immune response (12). His results showed that the synergistic effect of RT and immunotherapy has a time limit, and concurrent therapy is better than sequential therapy. In our study, the α-PD-1 mAb administered three days after 125 I RPI did not affect the e cacy of the combination therapy, possibly because the 125 I RPI and anti-PD-1 therapy have a longer synergistic time window. However, administration of 125 I RPI after dosing with three days of α-PD-1 mAb did reduce the e cacy. Our results are consistent with the study of Ahmed et al. where patients with non-small cell lung cancer (NSCLC) who received RT before or during the administration of anti-PD-1 had signi cantly longer survival than patients who received RT after anti-PD-1 therapy (28). This may be because radiation can promote the release of antigens, and this is conducive to the early initiation of anti-tumor immunity (31).
In addition, α-PD-1 mAb is only moderately effective and cannot signi cantly inhibit tumor growth. When 125 I particles are implanted, the tumor burden is too high, and 125 I RPI is a low-dose-rate RT. Dosage accumulation takes time, and this causes the tumor volume to be signi cantly inhibited within the experimental endpoint.
Clinical studies have found that single anti-PD-1/PD-L1 treatment does not have a curative effect in all cancer patients, and the objective response rate of NSCLC is < 20% (32). Studies have shown that anti-PD-1/PD-L1 therapy is bene cial in supporting su cient numbers of T cells in the TME (33). The immune promotion effect of RT can turn a drug-resistant population of anti-PD-1/PD-L1 into one that is bene cial (34). Clinical studies have proven that RT is an independent predictor of good prognosis for anti-PD-1/PD-L1 therapy in the treatment of NSCLC (35). Synergistic differences for anti-PD-1 treatment between different segmentation modes remain unknown. Here, we provide data on hyperfractionated brachytherapy combined with anti-PD-1/PD-L1 therapy. In terms of synergistic effects with immunotherapy, 125 I RPI has unique advantages over EBRT. First, it is highly conformable; during EBRT normal tissues also receive radiation, resulting in a decrease in radiosensitive lymphatic immune cells in these normal tissues, whereas normal tissues do not receive any radiation during brachytherapy. Also, the radiation radius of 125 I is only 1.7 cm, and the radiation intensity decreases as the distance increases, protecting normal tissues from radiation damage (36). Second, it has an ultra-long half-life; 125 I can provide ~ 180 days of irradiation, which can continuously promote tumor immunity. Anti-PD-1/PD-L1 treatment mostly uses disease progression or unacceptable toxicity as the withdrawal criteria. The time window for the synergistic effect between 125 I RPI and anti-PD-1/PD-L1 therapy is longer, resulting in longer anti-tumor immunity.
This study has some limitations. Although some immunophenotypes have been detected, additional intrinsic mechanisms need to be further explored. The effective radiation time of 125 I RPI is 180 days. An increase in the cumulative dose may enhance the curative effect. However, due to the limited survival time of tumor-bearing mice, it was not possible to monitor the synergistic effect of 125 I RPI and anti-PD-1 therapy over a longer time. We did not conduct any toxicity experiments, but no obvious toxicity was observed during our experiments. Also, our study has not been veri ed in other tumor models. We believe that 125 I RPI combined with anti-PD1 has broad applications in solid tumors, and especially in prostate cancer. Prostate cancer is less sensitive to anti-PD-1/PD-L1 treatment (37,38), but clinical trials have shown that activated T cells increase in the peripheral blood of prostate cancer patients gradually and continuously after 125 I RPI (39). 125 I RPI may improve the effect of prostate cancer immunotherapy.

Conclusion
This study shows that 125 I RPI combined with anti-PD-1/PD-L1 can signi cantly inhibit tumor growth and enhance anti-tumor immunity in mice. In addition, the e cacy is affected by the timing of 125 I RPI treatment. Although the potential advantages of 125 I RPI combined with anti-PD-1/PD-L1 treatment still need to be con rmed when compared with EBRT combined with anti-PD-1/PD-L1, our results for hyperfractionated brachytherapy combined with anti-PD-1/PD-L1 treatment are promising and lay the foundation for future clinical applications.