Estrogen receptor blockade and radiation therapy cooperate to enhance the response of immunologically cold ER+ breast cancer to immunotherapy.

BACKGROUND: Most patients with estrogen receptor positive (ER+) breast cancer do not respond to immune checkpoint inhibition (ICI); the tumor microenvironment (TME) of these cancers is generally immunosuppressive and contains few tumor-infiltrating lymphocytes. Radiation therapy (RT) can increase tumor inflammation and infiltration by lymphocytes but does not improve responses to ICIs in these patients. This may result, in part, from additional effects of RT that suppress anti-tumor immunity, including increased tumor infiltration by myeloid-derived suppressor cells and regulatory T cells. We hypothesized that anti-estrogens, which are a standard of care for ER+ breast cancer, may ameliorate these detrimental effects of RT by reducing the recruitment/ activation of suppressive immune populations in the radiated TME, increasing anti-tumor immunity and responsiveness to ICIs. METHODS: To interrogate the effect of the selective estrogen receptor downregulator, fulvestrant, on the irradiated TME in the absence of confounding growth inhibition by fulvestrant on tumor cells, we used the TC11 murine model of anti-estrogen resistant ER+ breast cancer. Tumors were orthotopically transplanted into immunocompetent syngeneic mice. Once tumors were established, we initiated treatment with fulvestrant or vehicle, followed by external beam RT one week later. We examined the number and activity of tumor infiltrating immune cells using flow cytometry, microscopy, transcript levels, and cytokine profiles. We tested whether fulvestrant improved tumor response and animal survival when added to the combination of RT and ICI. RESULTS: Despite resistance of TC11 tumors to anti-estrogen therapy alone, fulvestrant slowed tumor regrowth following RT, and significantly altered multiple immune populations in the irradiated TME. Fulvestrant reduced the influx of Ly6C+Ly6G+ cells, increased markers of pro-inflammatory myeloid cells and activated T cells, and augmented the ratio of CD8+: FOXP3+ T cells. In contrast to the minimal effects of ICIs when co-treated with either fulvestrant or RT alone, combinatorial treatment with fulvestrant, RT and ICIs significantly reduced tumor growth and prolonged survival. CONCLUSIONS: A combination of RT and fulvestrant can overcome the immunosuppressive TME in a preclinical model of ER+ breast cancer, enhancing the anti-tumor response and increasing the response to ICIs, even when growth of tumor cells is no longer estrogen sensitive.

Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy.

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.