Ionizing radiation normalizes the features of active breast cancer stromal fibroblasts and suppresses their paracrine pro-carcinogenic effects

Abstract

Background: Radiotherapy is an important therapeutic strategy for breast cancer patients through reducing the chances of recurrence and metastasis, which are fueled by cancer-associated fibroblasts (CAFs). Thereby, we addressed here the effect of various doses of X-rays on breast CAFs and their adjacent counterparts. Methods: We have used WST1 and annexin V-associated with flow cytometry to test the cytotoxic effects of X-rays. Immunoblotting and ELISA was used to assess the expression/secretion of various proteins. Immunohistochemistry was utilized to determine the level of β-galactosidase and Ki-67. Sphere formation assay was used to test the ability of breast cancer cells to form tumorspheres. Orthotopic tumor xenografts were also used to evaluate the effect of X-ray-treated breast stromal fibroblasts on breast cancer tumor growth in vivo. Results: Breast stromal fibroblasts showed high resistance to X-rays. While the low dose (5 Gy) inhibited cell proliferation and the active features of CAFs, the higher doses (16 and 50 Gy) promoted senescence. However, this was not accompanied by the senescence-associated secretory phenotype (SASP), but rather a reduction in the synthesis/secretion of various cancer-associated cytokines. Additionally, X-rays suppressed the features of active breast stromal fibroblasts, and their paracrine pro-carcinogenic effects. The ablative dose (16 Gy) inhibited the capacity of active stromal fibroblasts to promote the pro-metastatic processes epithelial-to-mesenchymal transition, the formation of cancer stem cells, as well as the growth of humanized orthotopic breast tumor xenografts. Conclusion: Together, these findings indicate that X-rays can normalize the features of active breast stromal fibroblasts through promoting senescence without SASP.

Keywords

• Breast cancer
• CAFs
• Ionizing radiation
• Senescence