TLR7 Agonist Imiquimod Improves the Therapeutic Antitumor Effect of High-Dose-Rate Brachytherapy.

1. Introduction
Radiotherapy (RT) is one of the cornerstones of cancer treatment. The main objective of curative RT is to achieve local tumor control successfully [1]. Radiotherapy activates inflammation and hypoxia in the tumor microenvironment. This leads to immunosuppression, creating new blood tumor vasculature and disease recurrence after radiation. Despite extensive advances, radio–resistance remains the main challenge hindering the effectiveness of radiation therapy. “Reinforcement” by tumor microenvironment is considered the seventh “R” of radiobiology. The tumor microenvironment (TME) is a pathological niche that contributes to tumor progression, metastasis, resistance to anticancer treatments, and survival [2]. The microenvironment consists of abnormal tumor blood vessels and immunosuppressive immune cells. Cancer cells manipulate this environment to evade immune action. Rapid tumor vasculature development leads to the formation of abnormal, dysfunctional blood vessels. Vasculature is characterized by branching, bulging, blind ends, defective basement membrane, pericyte coverage, poor blood perfusion, and hypoxia in the TME. Underoxygenation selected a robust and aggressive cancer subpopulation and suppressed antitumor immune reaction [3]. IR–induced DNA damage is lower in the absence of oxygen. Tumor hypoxia not only represents a hurdle for successful radiotherapy but also creates an immunosuppressive environment [4].
Pharmacologically induced normalization of the tumor vasculature is an interesting concept. The normalization process is highly dependent on correct dosing. Antiangiogenic strategies convert dysfunctional tumor blood vessels into a normal phenotype, resulting in increased oxygen delivery and tumor blood flow [4]. Increased tumor oxygenation should improve the therapeutic effects of radiotherapy. To overcome unfavorable processes within the TME, we attempted to combine the IMQ vessel normalizing dose with brachytherapy. Imiquimod (IMQ) is a TLR7 agonist that strongly activates innate and adaptive immunity. The FDA approves IMQ in a 5% topical formulation for the treatment of external genital warts, superficial basal cell carcinoma, and actinic keratosis. As we demonstrated, IMQ at a selective dose normalized tumor vasculature, both structurally (by reducing vessel tortuosity and increasing pericyte coverage) and functionally (by improving tumor perfusion). We outline IMQ repurposing as a vascular normalizing agent [5]. Imiquimod can also act as a radio–sensitizer and immune booster during radiotherapy in patients with melanoma [6]. As we mentioned, normalization of vascularization is one form of therapy that transforms the TME and improves the effectiveness of radiotherapy. We used brachytherapy (BT; interventional radiotherapy) at a dose that activated a robust immune response. As we previously showed, a dose of 10 Gy acts as “in situ” vaccination [7]. Brachytherapy is a form of contact radiotherapy that delivers a high dose to a tumor in a short time while keeping the whole–body radiation burden low [8]. The radiation source is close to or within the target volume [9]. A high dose per fraction and a reduction in overall treatment time can have greater antitumor effects and modulate the immune response [8].
Our study aimed to determine whether the addition of IMQ to anticancer therapy would overcome TME–mediated mechanisms of radiotherapy resistance. We verified the therapeutic efficacy of the proposed combination and observed a synergistic antitumor effect, primarily driven by tumor–infiltrating cytotoxic T lymphocytes. Our research results demonstrate the “reinforcement” of the tumor microenvironment, considered the seventh “R” of radiobiology. Appropriate conditions within the TME, as determined by IMQ, enhance the effectiveness of radiotherapy and lead to long–term tumor control. Our results indicate the direction for further development of combination therapies in the treatment of cancer patients. Knowledge gained may help design treatment regimens in the clinic in the future.

2. Materials and Methods

2.1. Cell Line
The murine melanoma IVISbrite™ B16–F10 Red F–luc tumor cell line (B16–F10; Perkin Elmer, Hopkinton, MA, USA) and 4T1 breast cancer cells (ATCC, Manassas, WV, USA) were maintained using RPMI (Biowest, Nuaillé, France) supplemented with 10% heat–inactivated fetal bovine serum (Eurx, Gdańsk, Poland) and 1% penicillin–streptomycin (Biowest). The cell cultures were passaged twice a week and cultured under standard conditions (37 °C, 5% CO2, 95% humidity).

2.2. Mice and Ethical Statement
Mice (eight–to–ten–week–old) C57Bl/6NCrl and BALB/cAnNCrl females (Charles River Breeding Laboratories, Wilmington, MA, USA) were bred in the Maria Sklodowska–Curie National Research Institute of Oncology, Gliwice Branch (Poland) in a HEPA–filtered Allentown’s IVC System (Allentown Caging Equipment Co., Allentown, NJ, USA). All efforts were made by qualified personnel to minimize animal suffering. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Committee on the Ethics of Animal Experiments of the Local Ethics Commission (Medical University of Silesia, Katowice, Poland) (Permit Numbers: 21/2020, 50A/2023, 50B/2023, 50D/2023, 21/2020/2024/P, 34/2025). Experiments on animals were conducted in accordance with the 3R rule and the ARRIVE guidelines (see Supplementary Materials). Mice were inoculated with tumor cells and were treated as described above. Mice whose tumor size exceeded 1 cm in any dimension (1.2 cm in individual cases) were sacrificed by cervical dislocation. After tumor cell injection, animal health was monitored daily (activity, appetite, behavior, and response to treatment). During this study, only a single animal in the control group displayed symptoms of suffering or met the termination criteria (weight loss > 20%, hunched posture, decreased activity/locomotion). During the experiments, we observed no side effects of the therapy (BCS ≥ 4) [10]. Procedures were terminated by cervical dislocation and tumor collection for analysis.

2.3. Inoculation of Animals and Therapeutic Agents
C57Bl/6NCrl mice or BALB/cAnNCrl females were injected subcutaneously with 2 × 105 B16–F10 or 4T1 tumor cells in 100 μL PBS, respectively. Growing tumors were measured with calipers, and tumor volumes were determined using the formula: volume = width2 × length × 0.52. Mice with well–developed tumors were treated with imiquimod and interventional radiotherapy (brachytherapy). Imiquimod (InvivoGen) was administered subcutaneously at a vascular–normalized dose 5 days before irradiation. A liquid formulation of the toll–like receptor 7 agonist imiquimod (IMQ; Imiquimod VacciGrade™ (R837), InvivoGen, Toulouse, France), dissolved in sterile water (acc. to the manufacturer’s specifications), was injected directly into tumors at a dose of 50 µg in 100 µL [5]. Control mice received sterile water directly into tumors.

2.4. Superficial Brachytherapy
Mice with well–developed tumors (~60–70 mm3 B16–F10 or ~40 mm3 4T1; n = 5 per group) were treated with contact interventional radiotherapy (brachytherapy). Brachytherapy was used at a dose of 10 Gy [7]. Superficial brachytherapy was performed with a Freiburg flap applicator (Elekta, Stockholm, Sweden), which was customized and placed directly into the tumor. The applicator was connected to a remote HDR afterloader with transfer tubes. After the treatment plan was ready, the irradiation started. The applicator was detached immediately after irradiation. The dose was planned to be specified at 2–4 mm from the applicator surface. The dose coverage was planned using Discovery RT (GE Healthcare, Chicago, IL, USA) computed tomography CT scans. Slice thickness was 1.25 mm. Spatial dose was calculated in the treatment planning system (OncentraBrachy, ELEKTA, Edmonton, AB, Canada). The distance was kept at least 14 cm to reduce the impact of radiation dose from neighboring mice. The time of irradiation was determined by the source activity (3–10 Ci). Irradiation was performed in the shielded therapeutic room with a high–dose–rate afterloader equipped with an iridium–192 radioactive source (Flexitron, Elekta, Stockholm, Sweden) in the Brachytherapy Unit, Maria Sklodowska–Curie National Research Institute of Oncology, Gliwice Branch (Poland). Mice were anesthetized by intraperitoneal injection of a mixture of 92,5 mg/kg ketamine (Ketanest 10 mg/mL) and 15 mg/kg xylazine (Sedazin 20 mg/mL) prior to irradiation.

2.5. Complete Blood Count Assessment
Mice’s blood withdrawal from the submandibular vein was conducted 8 days after brachytherapy. The blood was collected in EDTA–coated tubes. Parameters were assessed with the hematology analyzer scil Vet abc Plus+ (Mouse Research settings) (scil Vet abc Plus+, Horiba, Warsaw, Poland).

2.6. Cell Depletion and Depletion Status Analysis
For T lymphocytes and NK cells depletion, monoclonal antibodies (anti–CD8a clone: 53.6.72, anti–CD4 lymphocytes clone: GK1.5, or anti–NK cells clone: PK136; BioXCell, Lebanon, NH, USA) were injected intraperitoneally at a dose of 200 μg per mouse to maintain the depletion status. Depletion was monitored in peripheral blood samples and, at the end of the experiment, by staining CD4+, CD8+ T lymphocytes, and NK cells in blood, spleen, and tumors. To isolate T lymphocytes and NK cells, a single–cell suspension was prepared. Tumors were digested with collagenase II solution (500 U/mL; Gibco BRL, Grand Island, NY, USA). Red blood cells were lysed using ACK solution (Lonza, Morristown, NJ, USA). The obtained cell suspension was filtered using 70 μm and 40 μm cell strainers. Mononuclear cells were selected by centrifugation with Histopaque–1077 gradient (Merck, Darmstadt, Germany). The isolated cells were blocked with anti–mouse CD16/32 (clone: 93) antibody (BioLegend, San Diego, CA, USA) and stained with the following antibodies: anti–CD45 (clone: 30–F11), anti–CD8 (clone: YTS156.7.7), anti–CD4 (clone: RM4–4), and anti–CD49b (clone: DX5). Dead cells were stained using the viability dye DAPI (Merck). In flow cytometric analyses (BD FACSCanto II), gates separating negative from positive cells were based on fluorescence–minus–one (FMO) controls.

2.7. Statistical Analysis
The normality of distribution was verified by the Shapiro–Wilk test. The homogeneity of variance was verified using the Levene or Brown–Forsythe test. For variables meeting the conditions for parametric tests, analysis of variance (ANOVA) with post hoc Tukey’s HSD test was performed. For variables that did not meet the conditions for parametric tests, the Kruskal–Wallis test with multiple comparisons of mean ranks was performed. The Mantel–Cox test was used for the statistical evaluation of the mice’s survival. Variables are shown as mean ± SEM. p-value < 0.05 was considered statistically significant. All statistical comparisons were performed using Statistica 13 software.

3. Results

3.1. Brachytherapy of Subcutaneous Mouse Tumor Models
Brachytherapy is a radiation therapy technique in which a radioactive source is placed inside or in the proximity of a tumor [11]. It is recognized as an appropriate and effective method for treating non–melanoma skin cancers (NMSCs) using interstitial or surface techniques [12,13,14,15]. The latter method allows for delivery of the dose to the superficial tumors using specific applicators [11,12,16,17]. Serre et al. [18] indicate that, despite the development of brachytherapy in the clinic, preclinical studies are necessary to refine in vivo models to analyze the biological effects of BT, especially as an immunomodulatory systemic treatment. In this study, a superficial brachytherapy technique was used for irradiation with a commercial silicone flap surface applicator called the Freiburg flap. This applicator consisted of two parallel catheters 10 mm apart, surrounded by a 5 mm–radius silicone spacer. High–dose–rate brachytherapy (HDR BT) using the iridium–192 (Ir–192) isotope was applied. Mice were irradiated with a single dose of 10 Gy [7], specified to cover the entire tumor volume. The dose specification point was located at the distal part of the tumor visible on CT scans, 2–4 mm below the skin surface. The dose distribution was calculated using a brachytherapy planning system (Oncentra Brachy, ELEKTA, Edmonton, AB, Canada) based on computed tomography (CT). CT imaging was performed on a group of five mice, which were arranged as shown in Figure 1, and irradiated simultaneously. The mice were spaced at least 14 cm apart to minimize the effect of neighboring doses. Although this effect was minimal, it was taken into account when calculating the dose for each mouse. It is estimated that the contribution to the dose from neighbouring mice did not exceed 0.1 Gy. No additional shielding was used to separate the mice.

3.2. Imiquimod Sensitizing Brachytherapy In Vivo
We evaluated the therapeutic effect of a combination of selected doses in our previous research of imiquimod [5] and brachytherapy [7] in the treatment of murine B16–F10 melanoma (highly immunogenic, “hot” tumors) and 4T1 breast tumors (poorly immunogenic, “cold” tumors) [19]. We used a vascular–normalized dose (50 μg) of a liquid formulation of IMQ, which was subcutaneously injected into well–developed tumors with volumes of ~60–70 mm3 for B16–F10 (Figure 2a–d) or ~40 mm3 for 4T1 (Figure 2e–g). Brachytherapy was used at a dose of 10 Gy, which acts as an “in situ” vaccination leading to the development of a robust antitumor immune response [5]. To explore whether the antivascular/normalization effect of IMQ can be translated into a sensitizing effect on radiotherapy, tumor–bearing mice were treated with the indicated IMQ doses and irradiated. We observed long–term inhibition of murine tumor growth. The combination of imiquimod with brachytherapy most effectively inhibited murine melanoma tumor growth compared with the other groups (Figure 2a–d). Combined therapy had the greatest efficacy in delaying tumor growth, both in absolute and relative tumor volume, compared with monotherapy groups. We observed tumor growth inhibition with IMQ monotherapy or 10 Gy radiotherapy on the 22nd day of therapy. Tumor growth inhibition was 90% in IMQ + BT treatment compared to the control group (127 mm3 vs. 1425 mm3, respectively) and 70% compared to either treatment alone (127 mm3 vs. 454 mm3 IMQ vs. 464 mm3 BT alone). In breast cancer, tumor growth inhibition in mice treated with the combination therapy was greater than 95% compared with control mice on day 29 of therapy (6 mm3 vs. 402 mm3, respectively; Figure 2e–g). Complete tumor regression was observed in 20% of mice. After re–challenge with tumor cells, tumor growth was not observed. In long–term follow–up, we observed tumor progression after treatment with IMQ alone and large areas of necrosis in tumors from mice treated with brachytherapy alone. In mice treated with the IMQ + BT combination therapy, we observed long–term tumor growth control and prolonged survival.

3.3. The Contribution of Immune Cells to the Therapeutic Effect of Combining Imiquimod with Brachytherapy
We examined the contribution of immune cells to the antitumor therapeutic effect of the combination of imiquimod with brachytherapy. In this study, we focused on the melanoma model. Irradiation monotherapy caused leukocytopenia with decreased levels of lymphocytes, monocytes, and granulocytes in the blood of treated mice. After combined therapy, we observed decreases in platelet parameters and the total white blood cell count in the blood of treated mice. Combined therapy led to a systemic reduction in the number and percent of lymphocytes and monocytes in treated mice compared with other groups. Additionally, we reported increases in the percentage and count of eosinophils in the blood of mice. The peripheral blood eosinophil rate was 10–fold higher after combination therapy in mice than in the other groups (Figure 3a–c). Additionally, we observed a twice–increased level of tumor–infiltrating cytotoxic CD8+ T cells after combined therapy, compared to the control group (Figure 3d).
Next, we examined which subpopulation of immune cells, following combined therapy, plays a crucial role in inhibiting tumor growth. For this purpose, we used antibodies for specific immune cell subpopulation depletion from mice treated with the combination therapy (Figure 3d–g). Monoclonal antibodies (anti–CD8a, anti–CD4 lymphocytes, or anti–NK cells) were injected intraperitoneally at a dose of 200 μg per mouse on days 8 (1 day before IMQ administration), 10, 13, and 17. Depletion of natural killer cells or CD4+ T cells increased the effect of the combined therapy. After removal of NK cells or CD4+ T cells, we observed a reduction in tumor growth (by less than half and more than three–fold, respectively) in mice treated with the combination therapy. The tumor mass after depletion showed a more than two–fold reduction. After removal of CD8+ T cells from treated mice, we observed complete abolition of the therapeutic effect of imiquimod combined with brachytherapy. Depletion of CD8+ T cells increased tumor growth in treated mice by more than six–fold. These results indicate that cytotoxic CD8+ T cells play the most crucial role in the therapeutic effect of the combination of imiquimod with brachytherapy.

3.4. The Interconnected Imiquimod–Mediated Changes with Brachytherapy in the TME
The tumor microenvironment plays the leading role in tumor recurrence. Factors such as tumor size and density, tumor aggressiveness, hypoxia, the tumor’s ability to evade immune surveillance, and the quantity and quality of immune infiltrates are critical modifiers of antitumor therapeutic effect [18]. TME–mediated radio–resistance mechanisms, like increasing immunosuppression and hypoxia, lead to the recurrence of cancer after radiotherapy (Figure 4). Therefore, it is justified to use combination therapy: radiotherapy with drugs that modulate the tumor microenvironment. Normalization of tumor blood vessels and increased oxygen supply reduce tumor hypoxia, improving response to radiotherapy and preventing tumor recurrence after therapy [20]. Under the influence of treatment, the microenvironment should be reprogrammed into an antiangiogenic and immunostimulatory environment; in other words, one that inhibits tumor growth. The results of our studies indicate that an appropriate dose of IMQ normalizes vessels and activates the immune system, thereby inhibiting tumor growth and prolonging the survival time of irradiated mice.
In summary, combining a dose of IMQ that normalizes tumor vascularization with a dose of brachytherapy that activates an antitumor immune response induces a synergistic effect in long–term inhibition of tumor growth in treated mice. Our results clearly indicate that the use of drugs in combination with radiotherapy is justified, as they prevent the development of resistance mechanisms, including revascularization and immunosuppression, occurring in the tumor microenvironment.

4. Discussion
Brachytherapy (BT, interventional radiotherapy) is one of the radiation methods for treating patients with cancer. These irradiation techniques place radioactive sources directly in or near the tumor and deliver high doses to it [18,21]. We used contact–surface brachytherapy, in which an iridium–192 radioactive source was placed close to the cancer. Brachytherapy requires no additional margins around the clinical target volume. Sharp radiation dose gradients in BT have dosimetric advantages over conventional external–beam techniques. BT can achieve high tumor doses with decreased dose to critical organs. Additionally, the radiation source moves in tandem with the tumor during irradiation [22]. BT has greater potential to activate an immune response than a narrow dose of homogeneous external radiotherapy [18]. Despite the development of contact brachytherapy in the early 1900s [22], there is a need to refine radiation methods to enhance antitumor immune response [18]. Serre et al. [18] point out that, despite the development of clinical trials, there is a need for preclinical studies examining the specific biological effects involved in the systemic immunomodulatory effects of brachytherapy [18]. Our previous studies have shown that a dose of 10 Gy acts as an “in situ” vaccination, leading to the development of a robust antitumor immune response [7]. “In situ” tumor vaccination is a therapeutic strategy that converts a patient’s own tumor into a nidus for better presentation of tumor–specific antigens and for differentiation and stimulation of an antitumor–specific T cell immune response [23]. Patel et al. [23] reviewed that immune responses depended on the distance of cells from the BT source. The regions closest to the BT source showed enhanced immunogenic tumor cell death and release of tumor–specific antigens. Regions beyond the highest doses, including medium and high doses (8–12 Gy), induced double–strand breaks, DNA release, and activation of the cGAS/STING pathway [23].
Brachytherapy is an essential local therapy for solid tumors in clinical oncology. However, the limitations and the most significant benefits of the selected BT doses remain unknown. Single HDR fractions are used for skin cancers and partial breast treatment. Single–dose HDR BT is standard adjunctive therapy in prostate cancer. Radiotherapy causes cancer cell death through direct effects on DNA, but also alters the tumor’s immunological microenvironment [8]. BT appears to be more effective in inducing immunological phenomena than directed homogeneous external radiotherapy [18]. Our data showed the effect of radiation dose on the infiltration of immune cells (CD4+, CD8+ T cells, and NK cells) into melanoma tumors in treated mice. High doses (10 Gy or 15 Gy) effectively inhibited melanoma tumor growth. A dose of 10 Gy had the most significant impact on changes in tumor–infiltrating immune cells: it reduced tumor–associated macrophage levels and increased cytotoxic CD8+ T lymphocytes. Only a dose of 15 Gy increased NK cells in tumors of BT–treated mice [7]. The use of high–dose RT is a clinical trend in cancer treatment. Stewart et al. [8] emphasize the need to be cautious about using too high doses in hypofractionated regimens. Serre et al. [18] point out that the dose heterogeneity achieved with brachytherapy is a unique advantage compared to other RT modalities. The regions closest to the BT source receive the highest delivered dose. A range of BT radiation doses is delivered within the tumor volume, enabling local tumor control and protecting the immune system from immunosuppressive low doses that can affect nearby lymph nodes [18].
Brachytherapy is a promising method that can be used to test its combination with pharmaceutical immunomodulation therapies [18]. BT destroys cancer cells and also affects the tumor microenvironment (among others, tumor blood vessels and immune cells). In our research, we combined brachytherapy with imiquimod, an immunomodulating drug with antiangiogenic properties. TLR7 agonists localize to the endosomal membrane, triggering strong TH1–biased immune responses. They promote activation of CD8+ T cells. Unfortunately, the clinical use of TLR7 agonists is limited by the risk of an uncontrolled systemic immune response. Only imiquimod (IMQ) is U.S. Food and Drug Administration–approved [24]. The effect of IMQ is pleiotropic, stimulating both innate and adaptive immune responses. IMQ stimulates macrophage survival, DC maturation, and the synthesis of pro–inflammatory molecules, as well as the expression of costimulatory receptors on these antigen–presenting cells (APC). IMQ promotes Th1 skewing and increases the tumor–infiltrating T lymphocytes. A TLR7 agonist also reduces the levels of regulatory T lymphocytes and the CCL22 cytokine [5]. We used a single intratumoral injection, which minimizes systemic adverse responses. Mice showed no side effects following administration of the TLR7 agonist. Han et al. [25] synthesized cholesterolized liposomes containing a TLR7 agonist. 1V209–Cho–Lip effectively induced DCs activation and CD8+ T cells, and there were fewer adverse effects compared to the TLR7 agonist alone. In combination with 8 Gy of RT, it significantly inhibited tumor growth. Ota et al. [24] investigated a novel, systemically administrable TLR7–selective agonist, DSP–0509, which is being evaluated in a clinical trial. In vivo studies in combination with RT have demonstrated upregulation of CTL activity and inhibition of tumor growth. Yeh et al. [26] reported the first case series of three patients with unresectable cutaneous melanoma scalp metastases who were treated with topical imiquimod for 10–14 weeks in combination with brachytherapy (5 fractions of 6 Gy). BT precisely targets skin lesions with minimal brain irradiation. Scientists are seeking new clinical applications for IMQ. To confirm the antitumor efficacy of the IMQ + BT combination, we used a poorly immunogenic 4T1 breast cancer model. Adams et al. showed the safety and clinical efficacy of combining topical IMQ with radiotherapy in patients with metastatic breast cancer in a phase I/II trial. We observed prolonged survival in treated mice and complete tumor regression in 20% of them. After re–challenge with tumor cells, no tumor growth was observed, which indicates treatment–induced antitumor immune memory. We administered only a single normalizing dose of IMQ and observed an antitumor therapeutic effect. We are convinced that intratumoral administration of IMQ is a worthwhile alternative to prolonged topical administration.
There are conflicting data regarding the results of antiangiogenic drug regimens combined with IR. We realize that the therapeutic strategy we propose, combining IMQ with brachytherapy, used in the clinic, will not be the gold standard for all patients. Many patients demonstrate resistance to antiangiogenic drug therapy, and the treatment regimen will need to be tailored to the individual patient after tumor imaging. Telarovic et al. [4] noted that this is not a neo–adjuvant but rather a concomitant treatment. This results in an enhanced tumor response to irradiation. Antiangiogenic factors induce endothelial cell apoptosis and may thereby increase the sensitivity of tumor vessels to low–dose radiation, thereby improving the efficacy of radiotherapy. We administered BT based on the changes in the tumor after IMQ administration. However, each therapy must be optimized for the individual patient’s tumor. There are many proposed therapeutic strategies aimed at combining antiangiogenic therapy with immunostimulating agents. Our previous research has shown that a TLR7 agonist, which activates an antitumor response, also exhibits antiangiogenic properties at an appropriate dose and normalizes tumor blood vessels structurally and functionally. We observed significantly greater tumor growth inhibition and survival in treated mice. Normalization is very tricky—the drug itself, after increasing oxygenation, leads to rapid tumor growth. Therefore, chemotherapy or radiotherapy should be administered promptly. The combination leads to long–term control of tumor growth. Goedegebuure et al. [27] proposed the therapeutic triad comprising radiotherapy, antiangiogenic therapy, and immunotherapy. Optimization of the timing and dose schedule is essential for effective combination therapy with minimal adverse effects [27]. We have previously demonstrated that the doses of immunostimulating drugs, such as antiangiogenic and normalizing agents, should be examined. A single drug can act on both arms of the antitumor triad. In Jarosz–Biej’s work [5], we demonstrated that a 50 µg IMQ dose normalizes vessels and enhances the effectiveness of chemotherapy. Current research has shown that administering a single IMQ dose and normalizing vascularization in the long term increases the efficacy of radiotherapy in controlling tumor growth. Radiotherapy is not intended to eliminate tumors but to arrest their development. However, after some time, regrowth occurs. Therefore, it is necessary to combine radiotherapy with other forms of treatment to extend the time without progression of the cancer. It seems justified to combine normalization with radiotherapy to increase its effectiveness.
Normalizing the tumor microenvironment can reduce tumor density and promote immune cell infiltration [3]. Normalized tumor vasculature improves nutrient delivery and oxygen homeostasis, which sustains the proliferation and function of immune cells [3]. We focused on analyzing the immune cells involved in the therapeutic effect of the combination of imiquimod and brachytherapy. We observed a decrease in leukocyte numbers, accompanied by increases in blood granulocytes and eosinophils, in treated mice. The percentage of eosinophils in peripheral blood was 10–fold higher after combination therapy compared to the other groups. Carratero et al. [28] reported that activated eosinophils initiated processes in TME, including macrophage polarization, normalization of the tumor vasculature, and enhanced infiltration of CD8+ T cells, which promote tumor rejection. Lymphocytes are highly radiosensitive, and conventional tumor irradiation has been associated with lymphopenia. Up to 70% of patients undergoing conventionally fractionated RT develop radiation–induced lymphopenia. Lymphopenia has long–term effects and can persist for 4 to 8 years after treatment [29]. Our results showed that combined IMQ and BT therapy reduces lymphopenia compared with brachytherapy alone. Paquette and Oweida [29] noted that to improve tumor control and enhance the effectiveness of RT, it is necessary to activate and maintain an intact adaptive immune system during cancer therapy. The future direction of new RT regimens should be tailored to activate the antitumor immune response. The immune system, especially cytotoxic CD8+ T cells, plays a central role in this process. After depletion of the specific CD8+ T immune cell subpopulation in mice treated with the combination therapy, we observed a complete abolition of the therapeutic effect of imiquimod in combination with brachytherapy. Depletion of CD4+ T cells or natural killer cells enhances the impact of the combined therapy. These results indicate that cytotoxic CD8+ T lymphocytes play the most crucial role in the therapeutic effect of the combination of imiquimod with brachytherapy. Cytotoxic CD8+ T cells are attracted to the tumor site by the RT–induced chemokine release. These effector T cells recognize and destroy cancer cells. Transforming the tumor from “low intratumoral T cell infiltration type” to a “T cell inflamed phenotype” increased the effectiveness of therapy [7].

5. Conclusions
Combining a dose of TLR7 agonist imiquimod that normalizes tumor vascularization with a high–dose–rate (HDR) brachytherapy that activates an antitumor immune response induces a synergistic effect in long–term inhibition of tumor growth in treated mice. Our study results clearly support the rationale for combining drugs with radiotherapy to prevent the development of resistance mechanisms in the tumor microenvironment, including revascularization and immunosuppression. The direction of further development of antitumor combination therapies should be guided by tumor microenvironment changes observed during the treatment of oncological patients.