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

Background: Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer with poor prognosis. Immune-based therapy combined with conventional radiotherapy shows promise in suppressing TNBC growth and dissemination. The utilization of biocompatible immunoadjuvants for adjuvant radiotherapy of TNBC is attracting attention, although studies on this topic are still limited. Here we investigated the effects of a water-soluble immunoadjuvant, N-dihydrogalactochitosan [glycated chitosan (GC)] combining high-dose radiation (10 Gy) used in stereotactic body radiotherapy (SBRT) on TNBC 4T1 cells in vitro and in vivo. Methods: 4T1 cells were transduced with firefly luciferase reporter gene for in vivo monitoring of tumor progression after GC treatment and irradiation using the IVIS^® system. Fluorescent microscopy and Western blot analysis were used to investigate the effects of GC on cell morphology and immune related proteins, respectively. The cytokine antibody array was used to systematically compare the inflammatory molecules in 4T1 cells treated with or without GC. Results: While GC did not directly influence cellular morphology, it increased the expression of γ-H2AX associated with several inflammation regulatory factors including cGAS and programmed cell death ligand-1 (PD-L1) in a time-dependent manner. Furthermore, GC did not inhibit the growth of 4T1 tumors in vivo, although it changed the expression of several pro-tumor and anti-tumor cytokines. Pretreatment of GC enhanced irradiation induced γ-H2AX but not inflammation regulatory factors in 4T1 cells. For in vivo studies, the therapeutic efficacy of focusing high-dose radiation on 4T1 tumors was significantly better than GC treatment alone. A combination of GC and radiation showed comparable effect on tumor suppression with the radiation alone. Interestingly, the mice with the longest lifespan were observed in the group receiving the combined treatment. Conclusions: GC can influence specific inflammatory regulatory molecules and DNA damage marker γ-H2AX in TNBC cells. A combination of GC and high-dose radiation also exhibits the comparable efficacy with radiation alone using the syngeneic TNBC tumor model.