PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

Targeting CD73 with flavonoids inhibits cancer stem cells and increases lymphocyte infiltration in a triple-negative breast cancer mouse model.

Introduction: Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients, improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target, given its elevated expression on tumor, stromal, and specific immune cells, and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation, while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity, further suppressing anti-tumor immunity. While debulking the tumor mass, chemotherapy also enriches heterogenous cancer stem cells (CSC), potentially leading to tumor relapse. Therefore, drugs targeting both CD73, and CSCs hold promise for enhancing chemotherapy efficacy, overcoming treatment resistance, and improving clinical outcomes. However, safe and effective inhibitors of CD73 have not been developed as of now. Methods: We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry, RT-qPCR, CD73 activity, cell viability, tumorsphere formation, and other in vitro functional assays. For assessment of clinical translatability, TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. Results: We identified quercetin and luteolin, currently used as over-the-counter supplements, to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen, we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44highCD24low CSCs, and co-targeting CD73, YAP, and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore, triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Discussion: Conclusively, our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new, safe, and cost-effective therapeutic strategies for TNBC.

A New Functional Screening Platform Identifies Colistin Sulfate as an Enhancer of Natural Killer Cell Cytotoxicity.

Due to their crucial role in tumor immunity, NK cells have quickly became a prime target for immunotherapies, with the adoptive transfer of NK cells and the use of NK cell engagers quickly moving to the clinical stage. On the other hand, only a few studies have focused on small molecule drugs capable of unleashing NK cells against cancer. In this context, repurposing small molecules is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we developed a new platform to screen small molecule compounds based on a high-throughput luciferase-release cytotoxicity assay. We tested 1200 FDA approved drugs from the Prestwick Chemical Library, to identify compounds that increase NK cells' cytotoxic potential. We found that the antibiotic colistin sulfate increased the cytotoxicity of human NK cells towards cancer cells. The effect of colistin was short lived and was not observed when NK cells were pretreated with the drug, showing how NK cell activity was potentiated only when the compound was present at the time of recognition of cancer cells. Further studies are needed to uncover the mechanism of action and the pre-clinical efficacy of colistin sulfate in mouse cancer models.

Studying Cell Polarity Dynamics During Cancer Initiation Using Inducible 3D Organotypic Cultures.

Epithelial tissues are highly organized structures that are structured at both the cellular and tissue levels. Individual cells are characterized by an apical membrane facing a central lumen, and a basolateral membrane that contacts adjacent cells and the basement membrane. The maintenance of apical-basal polarity is crucial for maintaining epithelial homeostasis and is considered a barrier to carcinogenesis. Apical-basal cell polarity is compromised in many epithelial cancers, such as breast, lung, and prostate, and has been associated with disease progression. Three-dimensional (3D) organotypic cultures recapitulate the 3D tissue architecture and mechanical properties found in vivo. This chapter describes methods to establish 3D organoids from human cell lines or mouse primary cells with inducible oncogene expression in polarized epithelial structures to investigate mechanisms of tumor initiation, luminal filling, and growth. The method is versatile, and simple modifications can be made to study diverse cell/tissue types and oncogenes.