Exploring the Combined Effects of Tamoxifen and Radiotherapy, Regardless of Estrogen Receptor Status

Purpose/Objective(s)

Tamoxifen has been commonly used for the treatment of ER+ breast cancer with nanomolar affinity for the estrogen receptor. In recent years, the immunomodulatory effects of tamoxifen at micromolar concentration have gradually been discovered. However, its immunomodulatory action on anti-tumor was still ambiguous. Radiotherapy (RT) still faces primary and secondary radiation resistance which partially originates from the accumulation of tumor-associated macrophages (TAMs) with the M2 phenotype. Regulating the immune system may be an effective way to overcome radiotherapy tolerance. To date, the clinical effect of combining tamoxifen with RT has not been clearly defined. Our study aimed to uncover the synergistic effect of tamoxifen with RT and provide basic support for clinical applications.

Materials/Methods

Subcutaneous tumor models with three cell lines were adopted for certificating the synergistic effect of tamoxifen with RT. The infiltration status and function of immune cells among tumors are detected by flow cytometry. The tumor cell conditioned medium (CM) and CM after radiotherapy (RTCM) with or without tamoxifen were applied for stimulating bone marrow-derived macrophages (BMDMs) and RAW264.7 to TAMs. We performed RNA sequencing to detect the mechanism of the synergistic effect and completed the validation.

Results

In vivo experiments, it was gratifying that the anti-tumor effect of radiotherapy was all boosted in the combination group of RT and tamoxifen. In comparison, the synergistic action disappeared in immunodeficiency models. The infiltration of CD8+ T cells and CD86+ M1 TAMs was statistically higher in the combination group than the alone treatment group, while the CD206+M2 TAMs did not vary obviously. The proportion of Gzmb+ and TNF-α+ TAMs dramatically improved in the combination group. Furthermore, the proportion of activated CD8+ T cells and cytotoxic CD8+ T cells both remarkably increased in the combination group. After depleting TAMs with clodronate liposomes, the synergistic effect of RT with tamoxifen vanished. In vitro, the highest level of M1-related markers and the highest level of CD86+ macrophages both appeared in the combination group demonstrating the excellent combination effect of inducing TAMs M1 polarization. The RNA sequencing results revealed the mechanism by which RT combined with tamoxifen polarized TAMs to M1-type by activating the JNK/c-Jun pathway. The addition of JNK-IN-8, an inhibitor of JNK, supports the pivotal role of the JNK/c-Jun pathway in the process of M1 polarization induced by tamoxifen combined with RT.

Conclusion

All in all, we found RT combined with high-dose tamoxifen could significantly activate the JNK/c-Jun pathway, and then modulate the immune microenvironment, finally resulting in a stronger antitumor effect in vivo. The immunomodulatory function may open a new chapter for the application of tamoxifen.