Investigation of combination treatments using radiotherapy and stimulators of nucleic-acid sensing immune pathways in an organoid model of basal-like breast cancer

Abstract

The majority of triple-negative breast cancers (TNBC) are of the basal-like subtype, which has a poorer prognosis than non-basal TNBC. Few targeted therapies are approved for TNBC, and although immune checkpoint inhibitors have been licensed in both early and advanced breast cancer, the response rates in advanced disease are modest. Improved in vivo models and treatment approaches are required for basal-like breast cancer (BLBC). The first hypothesis tested in this thesis is that orthotopic implantation of organoids generated from tumors arising in BLG-Cre;Brca1f/f;p53+/- genetically engineered mice, will give rise to murine tumours with histological, genomic, and transcriptomic features of human BLBC. The second hypothesis tested is that in murine BLBC tumours, combination treatment with radiotherapy and stimulation of nucleic acid sensing immune pathways with generate immunogenic changes in the tumour microenvironment and prime tumours for immune checkpoint inhibition. A novel organoid-based experimental system was developed from murine mammary tumours generated in BLG-Cre;Brca1f/f;p53+/- mice, to test immuno-radiotherapy combination treatments in vivo. Validation of the novel organoid models showed heterogeneity between organoid lines and identified an optimal model, which demonstrated the basal-like phenotype and a high degree of similarity to human BLBC. Reovirus and STING agonists have previously been shown to activate nucleic acid-sensing activation pathways. In mice with bilateral organoid-derived mammary tumours, treatment of one tumour with reovirus and radiotherapy increased the CD8+ T cell infiltration in the treated and distant tumours compared with vehicle-treated controls (268 vs 31 cells/mm2 [p=0.02], and 219 vs 34 cells mm2 [p=0.03], respectively, at day 7 following treatment). Combination treatment with reovirus, radiotherapy and PD-1 checkpoint inhibition increased median survival compared with radiation monotherapy (32.5 vs 21.5 days [p=0.002]). Treatment using the STING agonist DMXAA with radiotherapy also increased CD8+ T cell infiltrates in irradiated and distant tumours compared with vehicle-treated controls (101 vs 14 cells/mm2 [p=0.04], and 82 vs 14 cells/mm2 [p=0.01], respectively, at day 13 following treatment). Combination treatment with the STING agonist STINGX, radiotherapy and PD-1 checkpoint inhibition generated complete responses in proportion of irradiated tumours and led to improved median survival compared with radiotherapy alone (34 vs 19 days [p=0.0002]). The findings in this thesis provide pre-clinical evidence that combination treatment with radiotherapy and stimulation of nucleic-sensing immune pathways may immunologically prime BLBC tumours for immune checkpoint blockade. These findings support further investigation of this therapeutic strategy in BLBC.