Investigating the STING pathway in the prostate cancer tumour immune microenvironment and radiosensitivity

Introduction & Objectives

The prostate cancer (PCa) tumour immune microenvironment (TIME) response to radiotherapy (RT) is a key area for research to optimise the anti-cancer immune response and improve clinical outcomes. Chronic inflammation is a driver of PCa development, however its role in tumour progression and radiosensitivity is poorly understood. The STING (STimulator of INterferon Genes) immune pathway is a key regulator of the immune response following viral infection and/or DNA damage. We investigated the role of STING, and its upstream cytoplasmic DNA sensor cGAS, in PCa tumorgenicity, modulation of the TIME, and response to RT using pre-clinical models.

Materials & Methods

Knockdown of cGas (cGas^KD) and Sting (Sting^KD) function in MyC-CaP and DVL3 PCa cells was generated using the Alt-R CRISPR system and validated using immunoblotting. Unmodified, cGas^KD and Sting^KD and MyC-CaP cells were injected subcutaneously into the flank of immunocompetent male FVB mice to assess tumorigenicity, tumour growth delay, and TIME effects (using flow cytometry, FACS) following treatment with 3 x 5 Gy fractionated RT administered at ~100 mm³ tumour volume.

Results

cGas^KD and Sting^KD MyC-CaP cells demonstrate increased sensitivity to a single 5 Gy fraction of RT in vitro, compared to unmodified cells. This enhanced radiosensitivity arising from cGas^KD and Sting^KD was not observed in DVL3 cells. cGas^KD and Sting^KD MyC-CaP flank tumours in syngeneic FVB mice demonstrated a reduced rate of tumour growth compared to control tumours. Further in vivo experiments demonstrated that cGas^KD and Sting^KD in MyC-CaP flank tumours enhanced the tumour growth delay effects of RT. FACS analysis of the TIME of MyC-CaP flank tumours demonstrated that RT results in fewer dendritic cells, total macrophages, M1-type macrophages, Treg-cells, CD4⁺ T-cells, and more CD11b⁺MHCII cells, monocytic MDSCs, B-cells, T-cells, compared to untreated tumours. RT treatment of cGas^KD MyC-CaP flank tumours resulted in fewer dendritic cells, total macrophages, B-cells, CD4⁺ cells and T-cells, and more M1-type macrophages, CD11b⁺MHCII cells, monocytic MDSCs, granulocytic MDSCs, NK cells, and CD8⁺ T-cells versus untreated cGas^KD tumours. RT treatment of Sting^KD MyC-CaP flank tumours resulted in fewer total macrophages, B-cells, CD4⁺ T-cells and T-cells, and more M1-type macrophages, CD11b⁺MHCII cells, monocytic MDSCs, granulocytic MDSCs, NK cells and CD8⁺ T-cells versus untreated Sting^KD tumours.

Conclusions

Taken together, these results suggest that the STING pathway modulates the sensitivity of PCa to RT, and influences the intrinsic composition of the TIME. The STING pathway is an attractive target for combination therapy alongside RT, and further research in enhancing the PCa anti-tumour response to RT is warranted.