Low-dose total body irradiation enhances systemic anti-tumor immunity induced by local cryotherapy

Strategies that restore the immune system's ability to recognize malignant cells have yielded clinical benefits but only in some patients. Tumor cells survive cryotherapy and produce a vast amount of antigens to trigger innate and adaptive responses. However, because tumor cells have developed immune escape mechanisms, cryotherapy alone may not be enough to induce a significant immune response. The mice were randomly divided into four groups: Group A: low-dose total body irradiation combined with cryotherapy (L-TBI+cryo); Group B: cryotherapy (cryo); Group C: low-dose total body irradiation(L-TBI); Group D: control group (Control). The tumor growth, recurrence, and survival time of mice in each group were compared and the effects of different treatments on systemic anti-tumor immunity were explored. L-TBI in conjunction with cryotherapy can effectively control tumor regrowth, inhibit tumor lung metastasis, extend the survival time of mice, and stimulate a long-term protective anti-tumor immune response to resist the re-challenge of tumor cells. The anti-tumor mechanism of this combination therapy may be related to the stimulation of inflammatory factors IFN-γ and IL-2, as well as an increase in immune effector cells (CD8+ T cells) and a decrease in immunosuppressive cells (MDSC, Treg cells) in the spleen or tumor tissue. We present unique treatment options for enhancing the immune response caused by cryotherapy, pointing to the way forward for cancer treatment.


Introduction
Cryoablation, an alternative to surgical resection, is widely used in the treatment of prostate cancer, esophageal cancer, renal cancer, breast cancer, and lung cancer (Tian et al. 2022; Thomsen et al. 2023;Tariq et al. 2020;Lin et al. 2023;Fine et al. 2021). With the development of image-guided technology, this modality of tumor treatment is becoming more and more popular (Mahnken et al. 2018). Cryotherapy can promote the formation of ice crystals in cells, directly causing physical damage to organelles and cell membranes without inducing defensive mutations in cancer cells (Erinjeri and Clark 2010;Baust and Gage 2005;Baust et al. 2019). It can also activate caspase-3 and BAX proteins to indirectly damage the mitochondria, thereby inducing tumor cell necrosis or apoptosis. Since the 1960s, cryotherapy has been known to have an aberrant immunoregulatory effect known as the "abscopal effect," and some studies have shown that it can affect or potentially eliminate metastatic tumors (Brok et al.
Yin Liao, Yao Chen and Shuya Liu have contributed equally to this work.

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2004; Annen, et al. 2022;Abdo et al. 2018). The necrotic cells persisted in the body after cryotherapy, which could trigger a robust immunological response. Tumor cell necrosis can lead to the release of the damage-associated molecular pattern (DAMP), including heat shock protein, S100 protein, non-protein components, and nucleic acids, as well as antigens and intracellular organelles, thereby promoting a significant increase in the levels of IL-1, TNF-α, NF-κB, and other inflammatory factors and chemokines (Kepp et al. 2020;Chen et al. 2023). It further stimulates the maturation of DC cells and the expression of costimulatory molecules CD80/86, as well as the activation of cytotoxic T lymphocytes (CTLs) to induce a systemic anti-tumor immune response (Brok et al. 2004;Kepp et al. 2020;Katzman et al. 2018). Cryotherapy alone, however, may not trigger an efficient immune response because tumor cells actively encourage the remodeling of their microenvironment to an immunosuppressive state (Shimizu et al. 2018;Davoodzadeh Gholami et al. 2017;Wu et al. 2022).
L-TBI within a particular range has been proven in studies to promote anti-cancer immunity or reverse the immunosuppressive status of the tumor microenvironment (Pandey et al. 2005). It can improve the "visibility" of immune attacks by activating immune cells and tumor cells to express the molecules and ligands required to initiate cytotoxic reactions, but it also suppresses a number of cells and cytokines associated with tumor-induced immunosuppression (Liu et al. 2010;Yang et al. 2014;Zheng et al. 2015). Usually, low-dose irradiation refers to a high linear energy transfer of ≤ 5 cGy or a low linear energy transfer of ≤ 20 cGy (Janiak et al. 2017). It has demonstrated a number of significant benefits, including simplified therapy, good patient tolerance, affordability, minor toxicity, and no harm to healthy tissues. L-TBI can improve the systemic immune response induced by hypofractionated radiation, as our team has previously shown (Liu et al. 2019).
Therefore, in this study, we demonstrated that L-TBI in conjunction with local cryotherapy can significantly inhibit lung metastasis in addition to effectively controlling local tumor regrowth. This combined therapy can considerably increase the survival period of mice by inducing a longterm protective anti-tumor immune response that can withstand the re-challenge of tumor cells. It implies that combining local cryotherapy with L-TBI may be a cutting-edge approach to treating cancer.

Methods
Cell culture and mouse models BALB/C mouse-derived mammary carcinoma 4T1 cells were obtained from the State Key Laboratory of Biotherapy of Sichuan University (Chengdu, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA), supplemented with 10% fetal bovine serum (FBS; Cellmax, Australia) and 1% penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA). Cell cultures were incubated at 37℃ with 5% CO 2 in a humidified incubator.
Female 6-8-week-old BALB/C mice weighing about 20 g were obtained from Chongqing Tengxin Biotechnology Co., LTD. (Chongqing, China). Mice were housed in separate specific pathogen-free (SPF) cages and allowed to eat and drink ad libitum. The ambient temperature was controlled between 20 and 25 °C to simulate the circadian rhythm. 4T1 mammary carcinoma cells (1.5 × 10 5 /ml) were subcutaneously injected into the right flank of each BALB/C mouse separately. This treatment time point was designated as day 0. Tumor size was monitored every 2 days, and tumor growth or regression was recorded. Tumors were measured with a vernier caliper and volume was determined using the formula: length x width 2 × 0.5 by two investigators independently. Tumors were selected for cryosurgery when they were 4-6 mm in diameter. Mice were sacrificed when tumors reached a volume of 1500 mm 3 .
For tumor rechallenge experiments, mice were challenged by subcutaneous injection of 4T1 cells (1.5 × 105/ml) in 0.1 ml of phosphate-buffered saline on the contralateral flank in the second experiment on day 19.

Cryotherapy and irradiation
Mice have been anesthetized with the aid of using intraperitoneal injection of chloral hydrate. Cryosurgery was performed using the direct contact liquid nitrogen method. The cotton swab was swiftly immersed in liquid nitrogen and used to contact the tumor surface, each time for about 10 s and lasted 1 min until the tumor formed an "ice ball". Then, the cycle was repeated after the ball melts. To guarantee optimum tumor cell death, a two-cycle 60-s freeze/thaw procedure was used. Mice were placed on a heating pad after the treatment to recover from anesthesia.
The mice were secured with a rubber band in a transparent cuboid self-made radiotherapy box. The 6-MV linear accelerator (Varian Clinac 600c, USA) was used for low-dose irradiation at a dose rate of 24 cGy/min, and the source-surface distance was maintained at 100 cm. Before irradiation, dose rates in the radiation field center and the middle plane were measured in the radiotherapy box, and the dose was verified by stacking thermoluminescent sheets near the tumor. L-TBI is defined in this study as whole-body irradiation at 0.1 Gy at a dose rate of 24 cGy/min.

Measurement of lung surface nodules
On day 26, the mice were sacrificed, and the lung tissue was collected and fixed with a 10% formalin solution. The pulmonary metastatic nodules were counted under an anatomical microscope and their diameters were measured. Metastatic nodules were divided into four grades based on their diameters: I < 0.5 mm, II 0.5 mm-1 mm, III 1 mm-2 mm, and IV > 2 mm. The numbers of pulmonary metastatic nodules were I × 1 + II × 2 + III × 3 + IV × 4. For histopathological examination, fixed tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) according to standard protocols. Microscopic analysis of all slides was performed with an optical microscope (Olympus Cor, Tokyo, Japan) linked to a computerized imaging system (Image-Pro Plus V6.0, Silver Spring, MD).

Flow cytometry analysis, ELISA measurements and immunohistochemistry
The tumor tissues were excised and homogenized in DMEM medium with 0.2% type IV collagenase, 0.01% hyaluronidase, and 0.002% DNase I (all enzymes from Solarbio Science, Beijing, China) for 40 min at 37 °C. Additionally, spleen tissues were dissected, ground, and filtered into a single-cell suspension using standard protocols. The blood cell lysate kits were used for removing red blood cells (BD Biosciences, CA, USA). The resulting single-cell suspension was stained with the fixable viability stain 780 before being tagged with the following antibodies as directed: CD45-PerCP antibody, CD11b-FITC antibody, Gr1-FITC antibody, CD11c-PE antibody, CD3-PerCP-Cy5.5 antibody, CD4-FITC antibody, and CD8-PE-Cy7 antibody. Stained samples were analyzed using a Beckman Coulter Gallios flow cytometer. All antibodies were purchased from BD, and the concentrations were the same as those of the antibodies used. All data measured by flow cytometry were analyzed using FlowJo software (version 10.0).
Tumor tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin before being cut into 4 µm thick sections. Baking, dewaxing, hydration, antigen repair and peroxidase blocking, adding antibody (CD4, CD8, Ki-67, and Foxp3), coloring, and re-staining were all performed. Finally, the slices were viewed under an optical microscope, and images were collected from five randomly selected fields under a 200X microscope. CD4, CD8, Ki-67, and Foxp3 positivity rates were counted by quantitative analysis with Image-Pro Plus 6.0 software (Media Cybernetics, USA), and data are expressed as mean ± standard error (SE). The percentage of positive cells = the number of positive cells / the number of total cells in this field.
On day 26, 0.5 ml of venous blood from the eyeball was collected, left to stand for 20 min at room temperature (20-25 °C), and then centrifuged (2000r, 20 min). The supernatant was collected as serum, inhaled under sterile conditions, and kept in a refrigerator at −20 °C for subsequent studies. According to the instructions for using the ELISA kit, the concentrations of IFN-γ, TNF-α, and IL-2 in serum were measured using the standard ELISA method.

Statistical analysis
SPSS18.0 (SPSS, Inc., Chicago, IL, USA) was used for data processing, and the enumeration data were expressed as mean ± standard error (mean ± SE). An independent sample t-test was used to compare the data between the two groups. One-way ANOVA was used to compare data from three or more groups. The Kaplan-Meier method was used for survival analysis. P < 0.05 was considered statistically significant. Fisher's exact test was used to analyze re-growth rates and re-challenges. All graphs were created with Graph-Pad Software Prism 7.0.

Cryotherapy combined with L-TBI can not only control tumor regrowth but also prolong the survival rate of mice
Cryotherapy can stimulate systemic anti-tumor immune responses by releasing large amounts of tumor-specific antigens. However, this immune response is limited. L-TBI can increase systemic antitumor effects outside of the radiation area induced by H-RT, as we have previously shown (Liu et al. 2019). Thus, in this study, we used BALB/C-derived mammary carcinoma 4T1 cells to develop a tumor model to test if local cryotherapy combined with L-TBI can trigger systemic antitumor effects. To this end, 4T1 cells were injected on the right flank of mice and then subjected to whole-body irradiation at 0.1 Gy when it reached 5 to 6 mm in diameter on about day 10 (Fig. 1A). The mouse tumors received cryotherapy two days later. Following that, tumor alterations and survival time were noted. L-TBI paired with cryotherapy resulted in a substantial reduction in relapses in mice (Fig. 1B). We found that the combination therapy effectively prolonged the survival of the mice, as shown in Fig. 1C.

Cryotherapy paired with L-TBI inhibited tumor lung metastasis
Breast cancer treatment has always faced the challenging issue of tumor metastasis. The 4T1 cells used in this study belong to a kind of tumor with significant invasion and metastasis that can rapidly spread to lung tissues (Pulaski 1 3 and Ostrand-Rosenberg 2001). As a result, on day 26, we calculated the lung tumor metastatic nodules. Although cryotherapy alone could only minimally limit lung metastasis as compared to the control group, the number of lung metastatic nodules in mice was dramatically reduced after being combined with L-TBI, as shown in Fig. 2.

Cryotherapy and L-TBI synergize to mediate the rejection of a subsequent 4T1 tumor challenge
We created a model system to test the anti-tumor immunological effects of the combination therapy to investigate if L-TBI paired with cryotherapy could stimulate a long-term protective anti-tumor immune response. Mice were challenged with a second injection of 4T1 cells on the opposite flank on day 19 (Fig. 3A). The growth of secondary tumors in mice following various treatments was observed. L-TBI-treated mice developed secondary tumors at the same rate as untreated mice, and tumor regrowth was seen in all mice. In contrast, combination therapy led to rejection of the second tumor (Fig. 3C). The control of distant metastases has always been the goal of cancer therapy. Here, we also evaluated the abscopal effects induced by different treatments. To model tumor metastasis, mice were re-inoculated with 4T1 cells subcutaneously on the opposite flank on day 2 (Fig. 3B). Similarly, tumors were treated at the aforesaid time. We monitor secondary tumor growth every two days. However, the results showed that cryotherapy combined with L-TBI did not affect contralateral tumor growth (Fig. 3D).

Combination therapy increased the level of serum IFN-γ and IL-2 and induced a higher proportion of CD 8 + T cells in the spleen
Cytokines play an important role in the anti-tumor immune response. To investigate the effect of L-TBI on the ability of cryotherapy to stimulate cytokines, we measured the production of IFN-γ, IL-2, and TNF-α in serum on day 26. As shown in Fig. 4, the combination therapy promoted a significant increase of cytokines IFN-γ and IL-2, which might have helped to enhance the immune response and control the growth and metastasis of tumors. Further, we investigated the effect of combination therapy on immune cells. Also on day 26, CD8 + T cell activation in the spleen was analyzed by flow cytometry. As shown in Fig. 5, the expression percentage of CD8 + T cells after cryotherapy combined with L-TBI was the highest. Cryotherapy, on the other hand, had no effect on CD8 + T cell expression when compared to the control group. This suggests that the combination therapy may successfully promote CD8 + T cell response and so play a role in tumor regrowth and metastasis control.
Myeloid-derived suppressor cells (MDSCs) are important members of the tumor immune milieu and they can effectively inhibit anti-tumor immunity (Kumar et al. 2016). On day 26, we also analyzed the expression of MDSCs by flow cytometry. The result showed that cryotherapy alone had no effect on the expression of MDSC cells, but combination therapy promoted the decrease of the expression of MDSC cells.

Combination therapy promoted the infiltration of CD8 + T cells and the reduction of MDSC cells in the tumor
In this study, we failed to observe the abscopal effect induced by combination therapy. We hypothesized that excessive tumor load in mice following bilateral tumor inoculation caused the death of the mice, which could have influenced the tumor volume record. As a result, we investigated the impact of various therapies on distant tumor cell proliferation and immune cell infiltration in tissues. Immunohistochemical staining was used on the 26th day to detect the expression ratios of Ki-67, CD4, CD8, and Foxp3 in distant tumor tissues of different groups. At the same time, a singlecell suspension was prepared for flow detection. Ki-67 is a well-known marker used to evaluate cell proliferation, and it is usually highly expressed in malignant tumor cells (Yang et al. 2018). The data showed the lowest percentage of Ki-67 expression in the combination therapy group, suggesting that the combination therapy may have inhibited the proliferative activity of tumor cells, although the tumor volume analysis did not show a statistically significant difference.
The immunohistochemical results of tumor tissues revealed that the proportion of CD8 + cells was highest in the combination therapy group (Fig. 6), showing that L-TBI might promote CD8 + cell infiltration in tumor tissues. Foxp3 is most abundant in CD4 + CD25 + regulatory T cells (Tregs) (Kim 2009), and Tregs are known to play a significant role in the immunosuppressive microenvironment of tumors (Nishikawa and Koyama 2021). In this study, both cryotherapy and combination therapy decreased the expression of Foxp3.
In addition, we detected the infiltration of CD8 + T cells and MDSCs in distant tumor tissues by flow cytometry (Fig. 7). The proportion of CD8 + T cells was higher in the cryotherapy group compared to the control group, but there was no difference in the expression of MDSC cells, indicating that cryotherapy can enhance anti-tumor immunity but may not ameliorate immunosuppression in tumor tissues. The proportion of CD8 + T cells in the combined treatment group was significantly increased, and the expression of MDSC cells was reduced. The findings suggest that L-TBI combined with cryotherapy can stimulate the infiltration of immune effector cells in the remote tumor microenvironment Fig. 6 The expressions of CD4, CD8, Foxp3 and Ki-67 in distant tumor tissues of each group were detected by immunohistochemical staining. A Immunohistochemical images of CD4, CD8, Foxp3 and Ki-67 in each group (× 200 times); B Expression analysis of CD4, CD8, Foxp3 and Ki-67 in each group. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns no statistical significance) and reduce MDSC cells, hence limiting tumor cell proliferation activity and providing long-term survival benefits to mice.

Discussion
Epidemiological and experimental studies have shown that moderate doses (0.1-2.0 Gy) or high doses (> 2 Gy) of ionizing radiation can stimulate acute inflammatory responses by promoting tumor cell death (Sampadi et al. 2022). However, radiation also damages surrounding normal organs and tissues, increasing the risk of secondary tumors (Janiak et al. 2017;Baselet et al. 2019). In contrast, such complications do not occur with low-dose exposure to low doses of ionizing radiation. L-TBI not only directly activates NK lymphocytes and other potentially cytotoxic anti-tumor cells (Pandey et al. 2005;Kojima et al. 2002), but also enhances the "visibility" of immune attacks by stimulating immune cells and tumor cells to express the molecules and ligands necessary to trigger cytotoxic reactions, such as stimulating the expression of CD2, CD28, B7, and NKG2 (Yang et al. 2014;Janiak et al. 2017). Moreover, it effectively inhibits various cells and cytokines associated with tumor-induced immunosuppressive responses, such as macrophages, Treg cells, IL-6, and IL-10, to reduce or reverse tumor-induced immunosuppression (Liu et al. 2010;Zheng et al. 2015;Safwat 2000). Therefore, in this study, low-dose 0.1 Gy whole-body irradiation was selected as the treatment modality and the effect of this treatment modality on cryotherapyinduced systemic anti-tumor immunity was investigated. Previous studies have shown that using two freezing cycles to expose cancer cells to −30 ~ −40 °C for 1 min can completely destroy the cells (Kepp et al. 2020). The "ice ball" formed by freezing, especially the second freeze-thaw cycle, is considered "fatal" (Wu et al. 2022). Therefore, we used (*P < 0.05, **P < 0.01, ****P < 0.0001, ns: no statistical significance) the model described above to observe the effects on immune responses in this experiment. We found that L-TBI combined with cryotherapy not only effectively controlled tumor growth, but also significantly suppressed lung metastases to achieve long-term tumor-free survival in a 4T1 breast cancer mouse model. This study further demonstrated that combination therapy effectively promoted the production of anti-tumor cytokines. IFN-γ is recognized to mediate antitumor immune response and exert toxic effects on tumor cells (Burke and Young 2019;Gerber et al. 2013;Takeda et al. 2001). IFN-γ promotes the differentiation of type 1 helper T cells (Th1) and CTL cells, and these cells can produce high levels of IFN-γ, thereby promoting the positive immune cycle (Jorgovanovic et al. 2020;Ikeda et al. 2002). The peripheral circulation of mice contained high levels of IFN-γ, following the combination of L-TBI and cryotherapy, which was much higher than that of cryotherapy alone, demonstrating that L-TBI promoted the immunological effect induced by cryotherapy. Th1-type inflammatory cytokines have a strong ability to stimulate an anti-tumor immune response. Among them, TNF-α can promote the maturation of dendritic cells (Miwa et al. 2012), and IL-2 contributes to the proliferation of anti-tumor effects, such as CD8 + T cells (Idriss and Naismith 2000;Bae et al. 2022). The level of serum inflammatory cytokines IL-2 increased considerably following combination therapy in this trial. The increased levels of the cytokines mentioned above may aid to amplify the immune response, ultimately limiting tumor lung metastasis.
Furthermore, when compared to single cryotherapy, combination treatment effectively promoted the generation of splenic DC cells and CD8 + T cells. It is generally known that immature DC cells, like sentinels, have a great potential to endocytose antigens in the peripheral circulation. When it recognizes antigens, it can differentiate into mature DC cells, migrate, and then deliver the antigens to T cells, thereby promoting anti-tumor immunity (Holmes et al. 1997;Kim et al. 1999;Wülfing and Günther 2015). Effector CD8 + T cells constitute the major force of anti-tumor immunity. Therefore, inducing the proliferation of CD8 + T cells that can kill cancer cells is crucial for controlling tumor growth. The proportion of CD8 + T cells in the spleen did not increase following cryotherapy in this experiment, while the proportion of DC cells and CD8 + T cells in the spleen increased significantly in the combination treatment. This indicates that L-TBI effectively enhanced the ability of cryotherapy-induced immune cells, which may be related to the increase of IL-2 and INF-γ in the circulation. MDSC cells are the precursors of granulocytes or macrophages, which can produce immunosuppressive molecules such as nitric oxide (NO), arsenicase-I, or reactive oxygen species (ROS) and significantly inhibit immune response (Parker et al. 2015;Gabrilovich 2017;Cicco et al. 2020). We discovered that while cryotherapy alone did not inhibit the formation of MDSC cells, the combination treatment did. The above findings demonstrate that combined treatment was more conducive to generating an immune response, suppressing tumor recurrence and metastasis, and significantly extending the survival time of mice. Furthermore, we confirmed that the immunological reaction elicited by the combined treatment had long-term immune memory since the majority of the mice resisted the challenge of tumor cell reinoculation following treatment.
Masaru et al. demonstrated that cryotherapy alone did not suppress distant tumor growth, but cryotherapy combined with immune adjuvant pretreatment of dendritic cell tumor injection significantly inhibited distant untreated tumor growth (Brok et al. 2006). In this experiment, we also investigated if L-TBI combined with cryotherapy could achieve distant tumor control in a mouse breast cancer metastasis model. Although no long-range effects induced by the combination therapy were observable, it promoted distant tumor microscopic proliferation and the infiltration of immune cells in tissues. In this experiment, we observed a significant decrease in the Ki67 index in the combination treatment group. This indicates that the combined treatment can inhibit the proliferative activity of distant tumor cells to some extent. Furthermore, both immunohistochemical staining and flow cytometry in the combined treatment group revealed a significant increase in the expression ratio of CD8 in tumor tissues, showing that the combined treatment enhanced the infiltration of CD8 + T cells into the tumor. When compared to cryotherapy alone, combined treatment had no effect on Foxp3 expression in tumor tissues. However, flow cytometry revealed a decrease in MDSC after combined therapy versus cryotherapy alone, suggesting that combined therapy may partially reverse immunosuppression in distant tumors. The studies presented above demonstrated that the combination therapy enhanced immune effector cell infiltration into the tumor and partially alleviated immunosuppression in the distant tumor microenvironment.
Finally, we hypothesize that cryotherapy promotes the proliferation and activation of DC cells and CD8 + T cells by releasing a large number of antigens and stimulating the production of inflammatory cytokines IFN-γ and IL2. DC cells also present antigens to enhance CD8 + T cell-mediated anti-tumor immune response, whereas L-TBI improves each response link and inhibits immunosuppressive MDSC infiltration in tumors, enhancing the immunological impact. Of course, cryotherapy in combination with immune checkpoint inhibitors, immunological adjuvants, and cell therapy is still being investigated, but L-TBI provides novel approaches for activating abscopal effects.