Subcellular localization of PD‐L1 and cell‐cycle‐dependent expression of nuclear PD‐L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy

The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40–55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition

Potential Roles of Tumor Cell- and Stroma Cell-Derived Small Extracellular Vesicles in Promoting a Pro-Angiogenic Tumor Microenvironment

Simple Summary In this review, we focus on the distinct functions of tumor-cell-derived small extracellular vesicles in promotion of angiogenesis and describe their potential as a therapeutic target for anti-angiogenic therapies. Also, we focus on extracellular vesicles derived from non-cancer cells and their potential role in stimulating a pro-angiogenic tumor microenvironment. The article describes the biogenesis of small extracellular vesicles and refers to their proteomic cargo components that play a role in promoting angiogenesis. Moreover, we explain how small extracellular vesicles derived from tumors and non-cancer cells can interact with recipient cells and alter their functions. We particularly focus on phenotypical and functional changes in endothelial cells, macrophages, and neutrophils that result in proangiogenic signaling. Extracellular vesicles (EVs) are produced and released by all cells and are present in all body fluids. They exist in a variety of sizes, however, small extracellular vesicles (sEVs), the EV subset with a size range from 30 to 150 nm, are of current interest. They are characterized by a distinct biogenesis and complex cargo composition, which reflects the cytosolic contents and cell-surface molecules of the parent cells. This cargo consists of proteins, nucleic acids, and lipids and is competent in inducing signaling cascades in recipient cells after surface interactions or in initiating the generation of a functional protein by delivering nucleic acids. Based on these characteristics, sEVs are now considered as important mediators of intercellular communication. One hallmark of sEVs is the promotion of angiogenesis. It was shown that sEVs interact with endothelial cells (ECs) and promote an angiogenic phenotype, ultimately leading to increased vascularization of solid tumors and disease progression. It was also shown that sEVs reprogram cells in the tumor microenvironment (TME) and act in a functionally cooperative fashion to promote angiogenesis by a paracrine mechanism involving the differential expression and secretion of angiogenic factors from other cell types. In this review, we will focus on the distinct functions of tumor-cell-derived sEVs (TEX) in promotion of angiogenesis and describe their potential as a therapeutic target for anti-angiogenic therapies. Also, we will focus on non-cancer stroma-cell-derived small extracellular vesicles and their potential role in stimulating a pro-angiogenic TME

Small extracellular vesicle-mediated bidirectional crosstalk between neutrophils and tumor cells

Neutrophils are the first line of defense against tissue injury and play an important role in tumor progression. Tumor-associated neutrophils (TANs) mediate pro-tumor immunosuppressive activity and their infiltration into tumors is associated with poor outcome in a variety of malignant diseases. The tumor cell-neutrophil crosstalk is mediated by small extracellular vesicles (sEVs) also referred to as exosomes which represent a major mechanism for intercellular communication. This review will address the role of neutrophil-derived sEVs (NEX) in reprogramming the TME and on mechanisms that regulate the dual potential of NEX to promote tumor progression on one hand and suppress tumor growth on the other. Emerging data suggest that both, NEX and tumor-derived sEVs (TEX) carry complex molecular cargos which upon delivery to recipient cells in the tumor microenvironment (TME) modulate their behavior and reprogram them to mediate pro-inflammatory or immunosuppressive responses. Although it remains unknown how the balance between the often conflicting signaling of TEX and NEX is regulated, this review is an attempt to provide insights into mechanisms that underpin this complex bidirectional crosstalk. A better understanding of the signals NEX process or deliver in the TME might lead to the development of novel approaches to the control of tumor progression in the future

The immunomodulatory ballet of tumour‐derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD‐L1 pathway in cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) is a global cancer burden with a 5‐year overall survival rate of around 50%, stagnant for decades. A tumour‐induced immunosuppressive microenvironment contributes to HNSCC progression, with the adenosine (ADO) pathway and an upregulated expression of inhibitory immune checkpoint regulators playing a key role in this context. The correlation between high neutrophil‐to‐lymphocyte ratio (NLR) with advanced tumour staging suggests involvement of neutrophils (NØ) in cancer progression. Interestingly, we associated a high NLR with an increased intracellular PD‐L1 localization in primary HNSCC samples, potentially mediating more aggressive tumour characteristics and therefore synergistically favouring tumour progression. Still, further research is needed to harness this knowledge for effective treatments and overcome resistance. Since it is hypothesized that the tumour microenvironment (TME) may be influenced by small extracellular vesicles (sEVs) secreted by tumours (TEX), this study aims to investigate the impact of HNSCC‐derived TEX on NØ and blockade of ADO receptors as a potential strategy to reverse the pro‐tumour phenotype of NØ. UMSCC47‐TEX exhibited CD73 enzymatic activity involved in ADO signalling, as well as the immune checkpoint inhibitor PD‐L1. Data revealed that TEX induce chemotaxis of NØ and the sustained interaction promotes a shift into a pro‐tumour phenotype, dependent on ADO receptors (P1R), increasing CD170high subpopulation, CD73 and PD‐L1 expression, followed by an immunosuppressive secretome. Blocking A3R reduced CD73 and PD‐L1 expression. Co‐culture experiments with HNSCC cells demonstrated that TEX‐modulated NØ increase the CD73/PD‐L1 axis, through Cyclin D‐CDK4/6 signalling. To support these findings, the CAM model with primary tumour was treated with NØ supernatant. Moreover, these NØ promoted an increase in migration, invasion, and reduced cell death. Targeting P1R on NØ, particularly A3R, exhibited potential therapeutic strategy to counteract immunosuppression in HNSCC. Understanding the TEX‐mediated crosstalk between tumours and NØ offers insights into immunomodulation for improving cancer therapies