Correspondence Open access Published: 18 December 2024 Kyungmin Kim1,2,3 na1, Kyung Oh Jung4 na1, Sera Oh1,2,3, Young-Hwa Kim1,3,10, Seok-Yong Lee1,2,3, Seongje Hong4, Su Han Cho6,8, Hyejin Kim7,8, Siyeon Rhee8,9, Gi Jeong Cheon1,3, Keon Wook Kang1,2,3, June-Key Chung1 & …Hyewon Youn1,3,5 Show authors

Experimental Hematology & Oncology volume 13, Article number: 120 (2024) Cite this article

Radiation therapy is widely used to treat breast cancer and is also an effective way to remove remaining breast cancer cells after surgical resection (mastectomy) to minimize the probability of cancer recurrence, especially in breast tissue or surrounding lymph nodes [1,2,3]. However, it has been reported that 3–15% of breast cancer patients experience recurrence within 10 years after treatment, and the prognosis in this case is relatively poor [4, 5]. Therefore, many studies have investigated and proposed tissue-associated or blood-associated biomarkers for predicting therapeutic responses or suggested therapeutic targets for improving prognosis after breast cancer treatment [6]. MiR-21, an onco-miR, has been clearly demonstrated to have higher expression in individuals with breast cancer [7,8,9,10]. However, the relationship between radiotherapy and miR-21 expression, as well as the impact of plasma miR-21 levels on the prognosis of breast cancer patients following radiotherapy, remains insufficiently explored.

In this study, we analyzed the relationship between radiation exposure and miR-21 levels in breast cancer patients using the TCGA database. Patients were categorized based on their radiotherapy status to assess potential differences in overall survival rates associated with miR-21 levels. Among patients who received radiotherapy (RT), those with high miR-21 levels had significantly poorer survival outcomes (p = 0.02, determined by log-rank test), while the overall survival rates of patients who did not receive RT were not affected by miR-21 expression (Fig. 1A). Additionally, a comparison of hazard ratios between the two groups-patients who received RT and those who did not-showed a significant difference, with high miR-21 expression correlating negatively with overall survival in the radiotherapy group (Fig. 1B-D). For experimental validation of the correlation between radiation intensity and miR-21 expression, we first evaluated the survival rate and viability of 4T1 mouse breast cancer cells under various radiation intensities. Employing the clonogenic assay, a well-established method for evaluating the survival rate of irradiated cancer cells, the number of colonies was counted 7–10 days post-irradiation. Exposure to 10 Gy radiation eliminated all cancer cells (Fig. 1E, F). Cell viability significantly decreased in a time-dependent manner up to 48 h after 10 Gy exposure (Fig. 1G). Irradiated 4T1 cells showed increased miR-21 expression, with levels rising by 1.55 ± 0.05 times at 5 Gy and 1.85 ± 0.01 times at 10 Gy compared to controls after 8 h, indicating that 10 Gy maintains elevated miR-21 levels longer (Fig. 1H). Using the EVmiRNA database, we also confirmed that exosomal miR-21 expression is upregulated in breast adenocarcinoma patients compared to healthy controls (Fig. 1I). Moreover, hsa-miR-21-5p expression was significantly higher in breast adenocarcinoma patients (Empirical p < 0.001) compared to other exosomal miRNAs, indicating that miR-21 is one of the most differentially expressed miRNAs in breast adenocarcinoma (Fig. 1J). For in vitro experiments, radiation intensities affect the exosome secretion. Exosome secretion increased significantly, with 5 Gy and 10 Gy resulting in 1.62 ± 0.15 and 2.20 ± 0.10-fold increases, respectively (Fig. 1K-M). The isolated exosomes were confirmed to express the exosomal marker proteins, CD63 and Alix, while lacking the cell marker protein, Calnexin. Their size was approximately 100 nm as determined by TEM and Nanosight analysis (Supplementary Fig. 1A). Additionally, exosomal miR-21 increased by 1.60 ± 0.29-fold after 5 Gy and 2.82 ± 0.28-fold after 10 Gy (Fig. 1N). We validated the transfer of exosomal miR-21 using a miR-21-Luciferase reporter system (Fig. 2A, B) and confirmed the uptake of exosomes labeled with Alexa 488-NHS (Fig. 2C). Bioluminescent signals in 4T1/miR-21-Luc2 cells were reduced by 78.28 ± 6.57% when treated with 10 Gy exosomes (Fig. 2D, E). To determine whether transfer of miR-21 could affect tumor proliferation and migration, 4T1 cells were incubated with 0, 5, and 10 Gy exosomes for 48 h. 4T1 cells incubated with 10 Gy exosomes exhibited 387.3 ± 13.42% higher proliferation than those treated with 0 Gy exosomes (Fig. 2F). The human breast cancer cell line MDA-MB-231 exhibited similar results, showing that 10 Gy irradiation led to an increase in exosomal miR-21 levels. Additionally, exosomes from cells exposed to 10 Gy irradiation significantly enhanced tumor proliferation (Supplementary Fig. 2). The survival rate of 10 Gy irradiated 4T1 cells was 2.96 ± 0.30 times higher when treated with 10 Gy exosomes (Fig. 2G, H), and xenograft tumors treated with 10 Gy exosomes grew significantly faster than those treated with 0 Gy (Fig. 2I). Moreover, migration assays showed a 1.75 ± 0.40-fold increase in 4T1 cells treated with 10 Gy exosomes compared to control (Fig. 2J, K), and wound-healing assay revealed that at 28 h, the wound distance was 24.61 ± 7.90 nm in 4T1 cells treated with 10 Gy exosomes and 61.37 ± 7.06 nm in 4T1 cells treated with 0 Gy exosomes (Fig. 2L, M). We confirmed alterations in the expression of proteins PTEN and TIMP-3, both associated with the miR-21 pathway, in 4T1 cells treated with exosomes (Supplementary Fig. 1B-D). This indicates that exosomal miR-21 can lead to activate downstream signaling, promoting tumor malignancy. We identified a potential role of exosomal miR-21 in influencing the tumor microenvironment by inducing M2 macrophage polarization (Supplementary Fig. 3). In addition, we confirmed exosomal miR-21 on fractionated radiation and validated the effects of miR-21 on tumor proliferation (Supplementary Fig. 4).

In conclusion, we have clarified the novel aspects of our study, which focuses on the effects of radiotherapy on miR-21 expression and its exosomal release in breast cancer cell lines and clinical data. We now emphasizes how different radiation doses impact exosomal miR-21 and its role in tumor proliferation and migration, as well as its potential implications for combination therapy with radiation.