Effects of N-dihydrogalactochitosan on the therapeutic efficacy of high dose radiation in a syngeneic triple negative breast tumor model

Introduction

Background

According to the reports of International Agency for Research on Cancer (IARC), a part of World Health Organization (WHO), breast cancer remains the most diagnosed cancer type in recent years, and an increase of 40% burden by 2040 has been predicted (1). Although the overall death rate of breast cancer has been declined by 43% from 1989 to 2020, the survival rate of triple-negative breast cancer (TNBC) is still the lowest (2). TNBC is known as a fast-growing and metastatic breast cancer with the worst prognosis (3). According to the Surveillance, Epidemiology, and End Results (SEER) database, the 5-year relative survival rates of TNBC with the stage at localized and distant spreading are 91% and 12%, respectively (4). Because TNBC lacks the expression of human epidermal growth factor receptor 2 (HER2), estrogen receptors (ERs), and progesterone receptors (PRs), most targeted treatments are not effective. It suggests that the prohibition of TNBC dissemination may increase the survival rate and extend the survival rate.

Rationale and knowledge gap

Radiotherapy is one of the conventional methods for cancer treatment. Stereotactic body radiotherapy (SBRT), also known as ultrahypofractionation radiotherapy, uses larger fraction size (dose/fx) and lower fraction number than external beam radiotherapy (EBRT). The advantage of SBRT is to deliver high radiation dose (10–15 Gy) to tumor lesions within a short timeframe (3–5 fractions) that would reduce the discomfort of patients and labors of clinicians, as well as lower the rate of postoperative complications and mortality (5). SBRT is commonly applied in early-stage lung cancer, prostate cancer and pancreatic cancer, or other cancer types that have metastasized to the lungs and liver. It has been reported that oligo-metastasis of breast cancer to liver could be treated by SBRT for effective tumor ablation accompanied by low systemic toxicity, based on several prospective trials (6). Because TNBC is highly metastatic, radiotherapy alone is insufficient to cure TNBC. Accumulated literatures and trials suggest that the therapeutic efficacy of radiation will be enhanced by combining immunotherapy (7-9). For instance, SBRT has been combined with the humanized antibody to programmed cell death protein 1 (PD-1), pembrolizumab, in a multi-institutional phase II trial (10). Improvement of patient survival and tumor control has also been reported by this combination (11). To increase the efficacy of SBRT on TNBC, development of different immune modulation agents should be interesting and important for this purpose.

N-dihydrogalactochitosan [glycated chitosan (GC)] is a biocompatible, multi-functional immunoadjuvant that has been used against various cancer types by stimulating the immune response in combination with ablation-induced cell killing and/or damage in vivo (12-19). The GC promoted cellular immunity related to cGAS-STING pathway mediated up-regulation of type I interferon (20). For modulation of tumor immunity, GC is injected into tumors after they were initially treated with different modalities, including photodynamic therapy (PDT) or photothermal therapy (PTT), radiofrequency ablation, high-intensity focused ultrasound (HIFU) (15-17,21). However, GC has not been combined with radiotherapy, a widely used modality for cancer treatment. Although radiotherapy is usually combined with various potent anticancer agents, it has not been combined with GC for in vivo study. We have previously combined GC and radiation in cultured 4T1 cells, and found that GC might be a potent radiosensitizer besides its immunoadjuvant capacity (22). An animal study is essential for demonstrating the combination of GC and radiation on TNBC therapy and is important for design of novel strategy in TNBC treatment. However, this preclinical study has not been processed.

Objective

In this study, we showed that GC did not cause obvious morphological change, but could induce several inflammatory factors including cGAS, cyclooxygenase-2 (COX-2) and programmed cell death ligand-1 (PD-L1) in 4T1 cells. GC alone did not influence directly the progression of primary 4T1 tumors in mice. We also demonstrated that pre-treatment of GC increased radiation-induced γ-H2AX, but post-treatment of GC did not lead to the same result. For the syngeneic 4T1 tumor model, combination of GC and high-dose radiation showed a similar effect with radiation alone, while the survival of several mice could be significantly extended by the combined treatment. The prospective of GC in combination with SBRT on TNBC treatment was discussed. We present this article in accordance with the ARRIVE reporting checklist (available at https://tro.amegroups.com/article/view/10.21037/tro-24-2/rc).

Methods

Cell lines

Murine 4T1 breast cancer cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in RPMI (Life Technologies Inc., Carlsbad, CA, USA) medium with 10% of fetal bovine serum, 50 µg/mL of penicillin-streptomycin, 2 mM of L-glutamine, and incubated at a 37 ℃ in a humidified incubator with 5% CO₂ and passaged every 48 hours. Cell images were visualized and acquired using the phase contrast microscope (Olympus CKX41, Tokyo, Japan) adapted a digital camera (OSTEC MC1200-MZ, Guangzhou, China).

Fluorescent staining of actin cytoskeleton

Tumor cells (1×10³) were cultured on coverslips overnight, followed by a fixation with 4% paraformaldehyde. Cells were then treated with 0.1% Triton X-100 and incubated with 0.5% fluorescine-conjugated phalloidin (Invitrogen Inc., Carlsbad, CA, USA) for 30 minutes. The coverslips were then rinsed with phosphate buffered saline (PBS) and stained with 1% 4',6-diamidino-2-phenylindole (DAPI; Invitrogen Inc.) in the dark for 10 minutes. The stained coverslips were placed on a slide and sealed with nail polish. Cells were visualized using a confocal fluorescent microscope (Leica DM 6000B, Wetzlar, Germany).

GC treatments

Cells were treated with 100 µg/mL GC (Immunophotonics, St. Louis, MO, USA) for different times. For in vivo studies, a 100-µL GC solution (10 mg/mL GC) was injected into the center and three random spots of the tumor as described previously with slight modifications (12). The control group was injected with PBS with the same procedure. For combining ionizing radiation (IR), cells were separated to pretreatment and posttreatment of GC, and then collected for protein extraction and Western blot analysis. For animal study, only pretreatment of GC was performed and radiation was delivered to tumor site immediately. The specification of X-ray machine is 160 kVp and 25 mA (RS 2000 Biological Research X-ray Irradiator; Rad Source Technologies, Inc., Suwanee, GA, USA), and the dose rate was set as 2.9 Gy/min according to the instrument’s instruction.

Western blot analysis and antibodies

Protein was extracted from cells using the protein lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 0.5% NP-40) with 2% phenylmethylsulfonyl fluorid (PMSF) and quantified using the Bio-Rad Protein Assay (Bio-Rad, Bio-Rad Laboratories Inc., Hercules, CA, USA). Protein was mixed with sampling buffer (250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulfate (SDS), 30% glycerol, 5% β-mercapitalethanol, 0.02% bromophenol blue), denatured with heating, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel was electrotransferred to a nitrocellulose membrane (BioTrace^TM NT, Pall, Port Washington, NY, USA) after electrophoresis, and the membrane was blocked with 4% milk in TBST buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 0.1% Tween20) for 2 hours. The membrane was incubated with primary antibody overnight, and followed by horseradish peroxidase (HRP)-conjugated secondary antibody (Millipore Co., Burlington, MA, USA). The membrane was rinsed with Western lightning plus-ECL (PerkinElmer Inc., Waltham, MA, USA) and the chemoluminescent signals were detected using the ImageQuant LAS-4000 gel imaging system (GE Healthcare, Buckinghamshire, UK). The band densities were quantified using ImageJ software (version 1.46). The primary antibodies used in this study included cofilin-1 (GTX102156), anti-destrin (GTX11071), PD-L1 (GTX31308), COX-2 (GTX100656), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GTX82899) (all above were purchased from GeneTex Inc., Alton Pkwy Irvine, CA, USA); anti-phospho-specific (ser3) cofilin (sc-271921, Santa Cruz Biotechnology Inc., Dallas, TX, USA); γH2AX (#9718), cGAS (#31659) (above were purchased from Cell Signaling Technology Inc., Beverly, MA, USA), and anti-β-actin (ab8227, Abcam Inc., Waltham, MA, USA).

Establishment of syngeneic TNBC tumor model

4T1 cells harboring firefly luciferase (fLuc) reporter gene was used for establishment of the syngeneic tumor model in immune competent female Balb/c mice as reported before (23). Mice were housing and fed by individually ventilated cage (IVC) system with controlled temperature and humidity. In brief, cells (1×10⁶ resuspended in 100 µL serum-free medium) were subcutaneously injected into the upper backs of 6 weeks old female Balb/c mice (National Laboratory Animal Center, Taipei, Taiwan). When tumors became palpable, their dimensions were measured using caliper twice a week. The tumor volume was estimated using the equation: volume = length (mm) × width² (mm²)/2. Tumor volume reached about 2,000 mm³ was set as experimental endpoint. The body weight of each tumor-bearing mouse was measured weekly. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University, Taipei, Taiwan (approval Nos. 1110107 and 1110601), in compliance with institutional guidelines for the care and use of animals.

In vivo bioluminescent imaging

Tumor-bearing mice were i.p. injected with 150 µL of 15 mg/mL D-luciferin (Gold Biotechnology, St Louis, MO, USA) for 15 minutes, and the animals were anesthetized with 2% isofluorane mixed with oxygen. The bioluminescent signals and 2D X-rays images were acquired using IVIS^® Lumina X5 Imaging System (PerkinElmer, Life and Analytical Sciences Inc., Waltham, MA, USA). Region of interests (ROI) were selected by circling the tumor regions or whole body, depending on the requirement of data interpretation. The unit of photon flux is set as photons/sec/cm²/sr.

Cytokine antibody array analysis

The C-series mouse cytokine antibody array C3 kit (Cat#AAM-CYT-3-8, Raybiotech Inc., Norcross GA, USA) was used to detect the cytokines in the tumorous samples following the manufacturer’s instructions. In brief, 250 µg protein extracted from excised tumors were mixed with blocking reagent and added to blocked cytokine antibody array membrane for 4 hours. The membrane was rinsed using wash buffer, and then incubated with biotinylated antibody cocktail at 4 ℃ overnight. After appropriate rinse process, the membrane was added with HRP-streptavidin and shaken at room temperature for 2 hours. After rinse, the membrane was developed using the chemoluminescent reagent and the signals were acquired using the ImageQuant LAS-4000 gel imaging system (GE Healthcare). The results were quantified using Image J software (version 1.46).

Statistical analysis

The data were analyzed using the statistic software GraphPad Prism (v6.0, GraphPad software, Boston, MA, USA). Each datum was represented as the mean of three independent experiments ± standard deviation. Data were analyzed with unpaired t-test. Two-way analysis of variance (ANOVA) was performed to compare the tumor burden and body weight after different treatments. The survival rate was determined using Kaplan-Meier method with a log-rank test. P<0.05 was regarded significance.

Results

Effects of GC on TNBC cells

We first examined the response of murine TNBC 4T1 cells to GC (Figure 1). The morphology did not change after cells were treated with GC up to 24 hours (Figure 1A). Using fluorescence microscopy and fluorescein-conjugated phalloidin staining, the fluorescence intensity of the actin cytoskeleton also did not change before or after GC treatment (Figure 1B). The actin-associated proteins cofilin-1 and destrin responsible for actin dynamics were not affected by GC treatment (24) (Figure 1C). However, we found that GC could induce the expression of cGAS, COX-2, PD-L1, and γ-H2AX, while the last two molecules also exhibited a time-dependent induction (Figure 1D).

Figure 1 Responses of 4T1 cells to the treatment of GC. (A) Visualization of cell morphology under the phase contrast microscope. There was no cell staining. Scale bar: 100 µm; (B) visualization fluorescein-conjugated phalloidin stained actin cytoskeleton using the fluorescent microscopy. Scale bar: 50 µm; (C) Western blot analysis of actin-associated proteins. GAPDH was used as the internal control; (D) effects of GC on the expression of cGAS, COX-2, PD-L1 and γ-H2AX. The blots were quantified using the densitometry and normalized to β-actin. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. GC, N-dihydrogalactochitosan/glycated chitosan; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; COX-2, cyclooxygenase-2; PD-L1, programmed cell death ligand-1.

Effects of GC on syngeneic TNBC tumor

We next established the syngeneic tumor model to investigate the effects of GC in vivo. The TNBC 4T1 cells harboring the fLuc reporter gene were s.c. implanted and imaged using the Lumina X5 system (Figure 2). Intratumoral injection of GC or PBS as control was performed when tumor size reached 200 mm³. The results of bioluminescent images and X-ray diagnostic images showed that GC did not affect the tumor growth (Figure 2A). The primary tumors at day 21, with or without GC treatment, were then excised and lyzed for analysis using the mouse cytokine antibody array (see Methods section). The semi-quantitative detection of 62 mouse cytokines revealed considerable alterations in the expressive levels of several cytokines, in accordance with the default cytokine table list outlined in the manufacturer’s manual (Figure 2B). For those cytokines changed equal or over 2 folds of mean levels, insulin-like growth factor binding protein 5 (IGF-BP-5) and keratinocyte chemoattractant (KC) were up-regulated, whereas eotaxin, a monokine induced by gamma interferon (MIG), was regulated on activation (Figure 2C). Regulated upon activation, normal T cell expressed and secreted (RANTES), thymus-expressed chemokine (TECK), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were down-regulated (Figure 2C). Current data suggest that GC alone would not directly affect the growth of primary tumors, although it can change the expression of several cytokines at tumor site.

Figure 2 Responses of syngeneic TNBC tumors injected with GC. (A) Bioluminescent imaging and X-ray imaging of 4T1 tumors with fLuc reporter gene. PBS and GC were intratumoral injected when tumor size reached 200 mm³. N=3; (B) mouse cytokine antibody array analysis of tumors after treatment for 21 days; (C) semi-quantification of all cytokines on the dot blots using densitometry. Each datum is the mean of duplicate dots for each cytokine. Red bars: control; orange bars: GC treatment. GC, N-dihydrogalactochitosan/glycated chitosan; POS, positive control; NEG, negative control; Axl, AXL receptor tyrosine kinase; BLC, B lymphocyte chemoattractant; CTACK, C-C motif chemokine ligand 27; GCSF, granulocyte colony-stimulating factor; GM-SCF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IGFBP, insulin-like growth factor binding protein; IL, interleukin; KC, keratinocyte chemoattractant; LIX, lipopolysaccharide-induced CXC chemokine; MCP, monocyte chemotactic protein; M-CSF, macrophage colony-stimulating factor; MIG, monokine induced by gamma interferon; RANTES, regulated upon activation, normal T cell expressed and secreted; SCF, stem cell factor; TARC, thymus and activation regulated chemokine; TCA-3, chemokine ligand 1 (CCL1); TECK, thymus-expressed chemokine; TIMP, tissue inhibitor of metalloproteinases; TNF, tumor necrosis factor; TPO, thyroid peroxidase; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; TNBC, triple-negative breast cancer; fLuc, firefly luciferase; PBS, phosphate buffered saline.

Responses of TNBC 4T1 cells to GC combining IR

We next investigated the effect of GC combining radiation on TNBC cells (Figure 3). A single treatment of GC or a single dose of X-rays (10 Gy), or their combination did not cause significant morphological change of TNBC 4T1 cells (Figure 3A). Next, we investigated the expression of cGAS, COX-2, PD-L1 and γ-H2AX based on the above conditions. We also compared the results of GC treatment before or after irradiation. The results showed that GC pretreatment followed by X-rays led to an increased level of γ-H2AX in cells, whereas the reverse order of treatments did not yield the same enhancement (Figure 3B). Interestingly, we also found that the inflammatory-induced COX-2 could be induced by GC or X-rays alone for 1 hour, but the immune checkpoint molecule PD-L1 could be induced only after 8 hours of each single treatment (Figure 3B). Moreover, induction of COX-2 and PD-L1 seemed stronger in cells exposed to X-rays followed by GC treatment, respectively (Figure 2B). These results suggest that pretreatment of GC may enhance radiation-induced DNA damage rather than induction of immune response, which may be modulated by GC treatment after X-rays exposure.

Figure 3 Responses of 4T1 cells to the combined treatment of GC and radiation. (A) Visualization and comparison of cell morphology with GC, radiation and combined treatment under the phase contrast microscope. There was no cell staining. Scale bar: 100 µm; (B) Western blot analysis of expression of COX-2, cGAS, PD-L1 and γ-H2AX. Each blot was quantified using densitometry and normalized by β-actin. GC, N-dihydrogalactochitosan/glycated chitosan; COX-2, cyclooxygenase-2; PD-L1, programmed cell death ligand-1.

Effects of combined GC and radiation on TNBC tumor growth

We next performed an in vivo experiment to investigate if GC can influence the radiation treatment of TNBC 4T1 tumors (Figure 4). The tumor was exposed to 10 Gy X-rays with or without the pre-intratumoral injection of GC, then the tumor volume was measured and in vivo bioluminescent imaging was performed, following the procedures in Figure 4A. Lead shielding was applied to protect the entire body of the tumor-bearing mouse, leaving only the subcutaneously implanted tumor exposed for irradiation (Figure 4B). A single dose of X-rays with or without the pre-treatment of GC showed similar effects on suppression of 4T1 tumors (Figure 4C). The body weights of tumor-bearing mice were not affected by high dose radiation with or without the GC treatment (Figure 4D). As use of 10 Gy is to mimic the high dose commonly applied for SBRT, current results suggest that GC has limited synergistic impact on the suppression of radiation treated tumors at the primary site.

Figure 4 Effects of GC and radiation on the growth of syngeneic TNBC tumor model. (A) A diagram of established syngeneic 4T1 tumor model used for the treatment of GC and/or radiation; (B) the photo represents the position of syngeneic TNBC tumor exposed for irradiation. The whole body was shielded by lead except the tumor lesion; (C) comparison of tumor growth curves in syngeneic 4T1 tumors treated with GC and/or IR; (D) comparison of body weight of tumor-bearing mice with different treatments. MEM, minimal essential medium; GC, N-dihydrogalactochitosan/glycated chitosan; IR, ionizing radiation; IVIS, in vivo imaging system; TNBC, triple-negative breast cancer.

In vivo bioluminescent imaging of TNBC tumor progression in response to GC and radiation treatment

We next investigated the metastasis of 4T1 syngeneic tumors harboring fLuc reporter gene in vivo using the bioluminescent imaging (Figure 5). The tumor progression with a time dependent manner was monitored by acquisition of photon flux emitted from luminescent tumor cells (Figure 5A). The photon flux of primary tumors (by selecting the ROIs) after different treatments was semi-quantified. It appeared that the photon signals of tumors were influenced by GC, which also enhanced the reduction of photon signals of tumors treated with high dose radiation (Figure 5B). However, the whole-body photon flux showed that tumor metastasis was not suppressed by a combination of GC and high dose radiation (Figure 5C). The combined GC and radiation treatment also did not significantly prolong the survival of the tumor-bearing mice, but the longest time of survival was observed in the group of combined treatment (Figure 5D). Taken together, current data showed that GC has limited effect on enhancing therapeutic efficacy of radiation treatment on TNBC, but individual responses to GC combined radiation treatment may influence the lifespan of tumor-bearing mice.

Figure 5 Effects of GC and radiation on the metastasis of syngeneic TNBC tumor model. (A) Bioluminescent reporter gene imaging of syngeneic 4T1 tumors with GC and/or IR treatments (all mouse number used for different treatments were shown in figure as is); (B) semi-quantification of photon flux at the positions of primary tumors with different treatments; (C) semi-quantification of photon flux for whole body of each tumor-bearing mice with different treatments; (D) comparison of survival rates of tumor-bearing mice with different treatments using the Kaplan-Meier Method with a log-rank test. ***, P<0.001. Exp., experiment; GC, N-dihydrogalactochitosan/glycated chitosan; IR, ionizing radiation; TNBC, triple-negative breast cancer.

Discussion

Key findings

Radiotherapy combining immunotherapy is the most focused strategy for cancer treatment contemporarily because of their syngeneic therapeutic effect. Targeting immune checkpoint is commonly used with radiotherapy that has been reported to induce PD-L1, a critical anti-tumor immunity associated molecule in both tumor cells and tumor microenvironment (25). Therefore, immunotherapy has become increasingly common in conjunction with radiotherapy for solid tumors. By 2019, however, there are only 7% (13 out of 185) of active immune checkpoint blockade (ICB) trials in combination with SBRT for the treatment of metastatic breast cancers (26). The objective response rate (ORR) of TNBC was moderate and was dependent on different ICB agents, but no significant complications were reported for a combination of SBRT and ICB for the treatment of TNBC (10,27,28). More clinical trials of ICB/SBRT combination are ongoing as this strategy is expected to provide optimistic outcomes for TNBC.

In this study, we have examined the effects of GC on cultured TNBC 4T1 cells, and found several phenomena that had not been reported previously. First, GC induces the expression of pro-inflammatory and molecules, including cGAS and COX-2 (Figure 1). Increase of cGAS was also accompanied by up-regulation of PD-L1, suggesting that GC may trigger signal pathways to regulate PD-L1 expression, such as the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway or the epithelial growth response receptor (EGFR) pathway (29-32). We also found that GC induced the expression of γ-H2AX (Figure 1). Although GC is not likely to induce DNA damage, GC may increase the cytosolic double stranded DNA (dsDNA) to trigger cGAS-STING pathway followed by increase of PD-L1. Cytosolic dsDNA can be generated from several origins, such as mitochondria or DNA rupture debris from cell nucleus (33). It is also possible to ingest the dsDNA from extracellular vesicles (34). The sources of dsDNA triggered by GC to induce the expression of γ-H2AX, as well as pro-inflammatory and immune responses is of interest to be further investigated.

Strengths and limitations

In syngeneic TNBC tumor model, GC did not greatly change the progression of primary tumor (Figure 2). However, GC treated tumors exhibited differential expression on several cytokines as shown in mouse cytokine antibody array (Figure 2). As GC alone did not directly suppress the primary tumor, the impact on these cytokines remains uncertain. The excised tumors may also contain other cell types and various cytokines in tumor microenvironment. For instance, KC is known to be expressed in dendritic cells, neutrophils and macrophages, but it also overexpresses in metastatic breast cancer cells (35,36). In addition, GC has been reported to stimulate the activity of macrophage, but it alone is insufficient to suppress tumor growth except through the combination of other ablation methods (37). While the up-regulated or down-regulated cytokines are associated with advance of breast cancer (38-40), GC may also influence the expression of these cytokines but compromise the growth of breast cancers. That is, it is likely that these cytokines can be a trigger of downstream actions when GC combined with other treatments. Radiation is also known to induce immune response in tumor microenvironment, which may be an important target for design of combination therapy (41). Combination of GC and high-dose radiation is speculated to influence the immune response and remodel tumor microenvironment. Because remodeling of tumor microenvironment is a complex process, it is difficult to define the precise regulatory effect of GC combining high-dose radiation on tumor-bearing mice. Regarding the expression of PD-L1, both GC and high-dose radiation exhibited the capacity to induce this immune checkpoint molecule after 8 hours of treatment (Figure 3B). A combination of GC and radiation could also induce the expression of PD-L1, but the level of induction did not stronger than the single treatment. Interestingly, pretreatment of GC exhibited slower PD-L1 expressive rate than posttreatment of GC when combining radiation, suggesting that pretreatment and posttreatment of GC may determine its function as a modulator for radiation and immune response, respectively. However, the regulatory effect of tumor microenvironment remodeling mediated by GC combining high-dose radiation needs to be further investigated in the future.

Comparison with similar researches

Through the evaluation of the tumors treated with radiation and GC, we have found that a 10 Gy radiation effectively suppressed tumor growth, irrespective of GC pretreatment (Figure 4). We have previously demonstrated that GC did not enhance the radiosensitivity of 4T1 TNBC cells up to 10 Gy in vitro, but it could enhance the radiosensitivity of liver metastatic 4T1 cells at high dose radiation (22). Current study using parental 4T1 cells for establishment of the syngeneic tumor model is partially consistent with previous findings. OM-174, an anti-tumor immunoadjuvant has been reported to enhance the radiosensitivity of tumor cells by interferon-γ secreted by murine splenocytes in vitro (42). Therefore, GC exhibiting dual functions as an immunoadjuvant and radiosensitizer is possible. However, the administration regime is important for determining the role of GC in cancer treatment. Additionally, GC has been shown to be synergistic with PDT, radiofrequency ablation and other modalities (15-17,19), the effect of GC combining SBRT remains intrigued and requires further investigation. Particularly, PDT generated cancer vaccines have been reported to be enhanced by GC both in vitro and in vivo (43). Therefore, it is believed that a combination of GC and PDT is to increase the therapeutic efficacy via enhanced generation of cancer vaccine. However, this strategy may be different from GC combining radiotherapy as GC is used as a radiation modulator rather than through the generation of cancer vaccine.

Explanations of findings

As GC can induce γ-H2AX, a combination of GC and radiation exposure should enhance the expression of γ-H2AX. Such an effect was only detected in cells pretreated with GC followed by radiation (Figure 3). Addition of GC immediately after radiation exposure did not increase the expression of γ-H2AX (Figure 3). As an immunoadjuvant, GC is injected into tumors that have been ablated by different physical methods (12,15,17,19). Interestingly, a previous report and current results suggest that GC would enhance the radiosensitivity when applied to cells prior to radiation exposure, and this is associated with the enhanced expression of γ-H2AX, a DNA damage marker (22). For the expression of COX-2 and PD-L1, the order of GC treatment with radiation did not dramatically influence the expression of these inflammatory/immune related molecules. While the exact mechanism requires further investigation, GC induced predeposition of γ-H2AX may has the potential to enhance cellular sensitivity to IR.

Implications and actions needed

We have used the bioluminescence imaging to assess the metastasis of 4T1 syngeneic tumors. It revealed that the addition of GC to radiation treatment increased tumor metastasis after 21 days of treatment (Figure 5). Due to the organic nature of GC, intratumoral injection of this compound followed by irradiation may potentially form intermolecular and/or intramolecular covalent bonds, leading to the formation of adduct structures that may affect tumor progression. OM-174, an anti-tumor immunoadjuvant has been reported to enhance the radiosensitivity of tumor cells by interferon-γ secreted by murine splenocytes stimulated by this compound in vitro (42). Radiosensitivity is defined by the death rate of cells exposed to radiation. Current data revealed that high dose radiation could show significant tumor suppression on 4T1 syngeneic tumors, while a combination with GC did not enhance the response of primary tumor. In addition, the control of tumor metastasis and survival rate were not improved by the combination of GC and high dose radiation. However, the longest survival of tumor-bearing mouse was still observed in the group of GC combined radiation treatment. It is interesting to investigate if GC combining radiation would exhibit the therapeutic efficacy on liver metastatic 4T1 cells in vivo in the future.

Conclusions

GC has been demonstrated as a biocompatible immunoadjuvant agent by combining laser therapy and has entered clinical trial previously (NCT03202446). However, GC needs to be injected into TNBC tumors before IR to raise its effect on radiosensitization in current syngeneic tumor model. This novel regime using GC to modulate the efficacy of radiotherapy may be important to assist the development of therapeutic strategy against TNBC. Additionally, GC can affect particular inflammatory regulatory molecules and DNA damage marker γ-H2AX within TNBC cells. The regulatory effect of combined use of GC and high-dose radiation on tumor microenvironment is interesting to be further studied to clarify the therapeutic mechanisms in vivo.

Acknowledgments

The authors thank Dr. Wei R. Chen, Dr. Tomas Hode and Dr. Samuel Siu Kit Lam for their precious comments and discussions on this study. They also thank Dr. Wei R. Chen for helping the editing works. They acknowledge Immunophotonics Inc. for providing IP-001(GC) for non-profit research purpose. The authors also thank the National Genomics Center for Clinical and Biotechnological Applications at the Cancer Progression Research Center, National Yang Ming Chiao Tung University, for the small animal imaging services. Additional support was provided by The National Core Facility for Biopharmaceuticals (NCFB), National Science and Technology Council.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tro.amegroups.com/article/view/10.21037/tro-24-2/rc

Data Sharing Statement: Available at https://tro.amegroups.com/article/view/10.21037/tro-24-2/dss

Peer Review File: Available at https://tro.amegroups.com/article/view/10.21037/tro-24-2/prf

Funding: This work was supported by Taoyuan General Hospital, Ministry of Health and Welfare (grant No. PTH111023), the Ministry of Science and Technology of Taiwan (grant No. MOST 111-2314-B-A49-037-MY3), the Ministry of Education, Higher Education SPROUT Project for Cancer Progression Research Center (No. 111W31101), and Cancer and Immunology Research Center (No. 112W31101). This work was also supported in part by the Stephenson Endowment, the University of Oklahoma.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tro.amegroups.com/article/view/10.21037/tro-24-2/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University, Taipei, Taiwan (approval Nos. 1110107 and 1110601), in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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