Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12–18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-β by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-β production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.

Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. , RT-TEX elicited tumor-specific CD8 T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. .

Cystathionine β-synthase (CBS) catalyzes metabolic reactions that convert homocysteine to cystathionine. To assess the role of CBS in human glioma, cells were stably transfected with lentiviral vectors encoding short hairpin RNA (shRNA) targeting CBS or a non-targeting control shRNA and subclones were injected into immunodeficient mice. Interestingly, decreased CBS expression did not affect proliferation in vitro but decreased the latency period prior to rapid tumor xenograft growth after subcutaneous injection and increased tumor incidence and volume following orthotopic implantation into the caudate-putamen. In soft agar colony formation assays, CBS knockdown subclones displayed increased anchorage-independent growth. Molecular analysis revealed that CBS knockdown subclones expressed higher basal levels of the transcriptional activator hypoxia-inducible factor 2α (HIF-2α/EPAS1). HIF-2α knockdown counteracted the effect of CBS knockdown on anchorage-independent growth. Bioinformatic analysis of mRNA expression data from human glioma specimens revealed a significant association between low expression of CBS mRNA and high expression of angiopoietin-like 4 (ANGPTL4) and vascular endothelial growth factor (VEGF) transcripts, which are HIF-2 target gene products that were also increased in CBS knockdown subclones. These results suggest that decreased CBS expression in glioma increases HIF-2α protein levels and HIF-2 target gene expression, which promotes glioma tumor formation. Implications CBS loss of function promotes glioma growth.

Introduction Inflammatory breast cancer (IBC) is an aggressive and rare cancer with a poor prognosis and a need for novel targeted therapeutic strategies. Preclinical IBC data demonstrates strong activation of the PI3K/mTOR and JAK/STAT pathways, expression of inflammatory cytokines and tumor associated macrophages (TAMs). Methods Archival tumor tissue from three disease types (IBC treated with neoadjuvant chemotherapy (NAC) (n=45); invasive ductal carcinoma (IDC) treated with NAC (n=24; ‘treated IDC’); and untreated IDC (n=27; ‘untreated IDC’)) was analyzed for the expression of biomarkers pS6 (mTOR), pJAK2, pSTAT3, IL6, CD68 (monocytes, macrophages) and CD163 (TAMs). Surrounding non-tumor tissue was also analyzed. Results Biomarker levels and surrogate activity by site-specific phosphorylation were demonstrated in the tumor tissue of all three disease types but were highest in IBC and treated IDC and lowest in untreated IDC for pS6, pJAK2, pSTAT3 and IL6. Of 37 IBC patients with complete biomarker data available, 100% were pS6 positive and 95% were pJAK2 positive. In non-tumor tissue, biomarker levels were observed in all groups but were generally highest in untreated IDC and lowest in IBC, except for JAK2. Conclusions IBC and treated IDC display similar levels of mTOR and JAK2 biomarker activation, suggesting a potential mechanism of resistance after NAC. Biomarker levels in surrounding non-tumor tissue suggest that the stroma may be activated by chemotherapy and resembles the oncogenic tumor-promoting environment. Activation of both pS6 and pJAK2 in IBC may support dual targeting of mTOR and JAK/STAT pathways, and the need for prospective studies to investigate combinatorial targeted therapies in IBC.

BackgroundGlioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion.MethodsIn order to perform the studies, we employed optimal cell culture methods and hypoxic conditions, lentivirus-mediated knockdown of protein expression, Western Blot analysis, migration assays and immunoprecipitation. We determined statistical significance by unpaired t-test.ResultsIn this report, we show that U87MG, LN229 and LN308 glioma cells express CXCR7 and that exposure to hypoxia upregulates CXCR7 protein expression in these cell lines. CXCR7-expressing U87MG, LN229 and LN308 glioma cells migrated towards stromal-derived factor (SDF)-1α/CXCL12 in hypoxic conditions in the Boyden chamber assays. While shRNA-mediated knockdown of CXCR7 expression did not affect the migration of any of the three cell lines in normoxic conditions, we observed a reduction in the migration of LN229 and LN308, but not U87MG, glioma cells towards SDF-1α in hypoxic conditions. In addition, knockdown of CXCR7 expression in LN229 and LN308 glioma cells decreased levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. Inhibiting CXCR4 in LN229 and LN308 glioma cells that were knocked down for CXCR7 did not further reduce migration towards SDF-1α in hypoxic conditions and did not affect the levels of phosphorylated ERK1/2 and Akt. Analysis of immunoprecipitated CXCR4 from LN229 and LN308 glioma cells revealed co-precipitated CXCR7.ConclusionsTaken together, our findings indicate that both CXCR4 and CXCR7 mediate glioma cell migration towards SDF-1α in hypoxic conditions and support the development of therapeutic agents targeting these receptors.