Single-cell transcriptomics reveal the microenvironment landscape of perfluorooctane sulfonate-induced liver injury in female mice.

Epidemic and animal studies have reported that perfluoroalkyl and polyfluoroalkyl substances (PFASs) are strongly associated with liver injury; however, to date, the effects of PFASs on the hepatic microenvironment remain largely unknown. In this study, we established perfluorooctane sulfonic acid (PFOS)-induced liver injury models by providing male and female C57BL/6 mice with water containing PFOS at varying doses for 4 weeks. Hematoxylin and eosin staining revealed that PFOS induced liver injury in both sexes. Elevated levels of serum aminotransferases including those of alanine aminotransferase and aspartate transaminase were detected in the serum of mice treated with PFOS. Female mice exhibited more severe liver injury than male mice. We collected the livers from female mice and performed single-cell RNA sequencing. In total, 36,529 cells were included and grouped into 10 major cell types: B cells, granulocytes, T cells, NK cells, monocytes, dendritic cells, macrophages, endothelial cells, fibroblasts, and hepatocytes. Osteoclast differentiation was upregulated and the T cell receptor signaling pathway was significantly downregulated in PFOS-treated livers. Further analyses revealed that among immune cell clusters in PFOS-treated livers, Tcf7+CD4+T cells were predominantly downregulated, whereas conventional dendritic cells and macrophages were upregulated. Among the fibroblast subpopulations, hepatic stellate cells were significantly enriched in PFOS-treated female mice. CellphoneDB analysis suggested that fibroblasts interact closely with endothelial cells. The major ligand-receptor pairs between fibroblasts and endothelial cells in PFOS-treated livers were Dpp4_Cxcl12, Ackr3_Cxcl12, and Flt1_complex_Vegfa. These genes are associated with directing cell migration and angiogenesis. Our study provides a general framework for understanding the microenvironment in the livers of female mice exposed to PFOS at the single-cell level.

Galectin-9 blockade synergizes with ATM inhibition to induce potent anti-tumor immunity.

Although current cancer immunotherapies that target PD-1/PD-L1 immune checkpoint to reinvigorate exhausted T cells have achieved impressive clinical outcomes, only a small proportion of patients respond. New therapeutic targets are therefore needed to be identified to further unleash the anti-tumor potential of T cells and benefit more patients. Galectin-9 (Gal-9), initially identified as a ligand for TIM-3 to induce T cell death, acts as an immunosuppressive regulator in the tumor microenvironment (TME) but its potential as a therapeutic target remains largely elusive. Here we show that antibody neutralization of Gal-9, in combination with inhibition of Ataxia telangiectasia mutated (ATM), a kinase essential for DNA damage response (DDR), is a promising modality for cancer immunotherapy. Genetic depletion of ATM in tumors markedly potentiated anti-Gal-9 therapy in a syngeneic mouse model. Mechanistically, ATM inhibition greatly upregulated Gal-9 expression and secretion in a variety of human and murine tumor cells via the cGAS-STING-interferon ß (IFNß) innate immune pathway. Combination of Gal-9 inhibition with AZD1390, a selective ATM inhibitor currently evaluated in clinical trials, significantly suppressed tumor growth and prolonged survival in multiple syngeneic mouse models, including the poorly-immunogenic LLC lung tumors that do not respond to PD-1/PD-L1 blockade, concomitant with increased T cell infiltration. These results reveal Gal-9 induction via STING/IFNß signaling as an important mechanism mediating tumor immune escape that could be targeted for cancer immunotherapies, and unveil a novel anti-Gal-9-based combination strategy for cancer immunotherapies in a wide variety of malignancies, including those resistant to PD-1/PD-L1 blockade.

PD-L1 deficiency sensitizes tumor cells to DNA-PK inhibition and enhances cGAS-STING activation.

Immunotherapies that block PD-L1/PD-1 immune checkpoint proteins represent a landmark breakthrough in cancer treatment. Although the role of PD-L1 in suppressing T cell activity has been extensively studied, its cancer cell-intrinsic functions are not well understood. Herein, we demonstrated that PD-L1 is important for the repair of DNA damage in cancer cells. Mechanically, depletion of PD-L1 led to the downregulation of the critical molecules involved in the homologous recombination (HR) repair pathway, such as ATM and BRCA1, but did not obviously affect the non-homologous end joining (NHEJ) pathway. Notably, PD-L1 silence sensitized cancer cells to chemotherapy agents and the inhibitor of DNA-PK, which is an important kinase for NHEJ. Furthermore, PD-L1 depletion potentiated DNA damage-induced cGAS-STING pathway and induction of IFNß. The regulation of DNA repair and cGAS-STING pathway by PD-L1 represents its connection with innate immunity that can be exploited to enhance the efficacy of existing immunotherapy. Our findings thus expand the focus of PD-L1 from tumor antigen-specific CD8+ T cells to innate immunity, and support targeting tumor-intrinsic PD-L1 combined with DNA-PK inhibition for tumor eradication, through promoting synthetic lethality and innate immune response.

Ferroptosis: a promising target for cancer immunotherapy.

Ferroptosis is a recently recognized type of programmed cell death and emerges to play an important role in cancer biology and therapies. This unique form of cell death, characterized by iron-dependent lipid peroxidation, is exquisitely regulated by the cellular metabolic networks such as lipid, iron and amino acid metabolism. The sensitivity to ferroptosis varies among different tumors. Recent evidence reveals that triple-negative breast cancer (TNBC), a highly aggressive disease with limited effective targeted therapies is particularly vulnerable to ferroptosis inducers, suggesting this new form of non-apoptotic cell death as an attractive target for the treatment of the "difficult-to-treat" tumor. Intriguingly, ferroptosis has recently been implicated to be involved in T cell-mediated anti-tumor immunity and affect the efficacy of cancer immunotherapy. Better understanding of this ferroptotic cell death will shed light on the discovery of novel combination therapeutic strategies for cancer treatment. Herein, we provide an overview of the key hallmarks of ferroptosis, use TNBC as a model to characterize the regulation of ferroptosis in cancer, and highlight ferroptosis-modulating combination therapeutic strategies in the context of cancer immunotherapy.